Name: Anti-S6K1 antibody [E343]
SKU: ab32529
Rating: 1 (1 reviews)

Rabbit Recombinant Monoclonal KS6B1 antibody. Suitable for IP, WB, ICC/IF, Flow Cyt (Intra), IHC-P and reacts with Mouse, Human, Rat samples. Cited in 96 publications.

Images

Western blot - Anti-S6K1 antibody [E343] (AB32529), expandable thumbnail

Publications

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 50% Glycerol (glycerin, glycerine), 49% PBS, 0.05% BSA
Form: Liquid
Clonality: Monoclonal

Immunogen

The exact immunogen used to generate this antibody is proprietary information.

Reactivity data

Select an application

Product promiseTestedExpectedPredictedNot recommended

	IP	WB	ICC/IF	Flow Cyt (Intra)	IHC-P
Human	Tested	Tested	Tested	Tested	Tested
Mouse	Tested	Tested	Tested	Tested	Tested
Rat	Expected	Tested	Tested	Tested	Tested

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/110	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Species Human	Dilution info 1/110	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Expected
Expected

Species	Dilution info	Notes
Species Rat	Dilution info Use at an assay dependent concentration.	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/5000 - 1/10000	Notes For Rat and Mouse samples 1/500 dilution has only been tried. We have not tested if similarly to Human samples a lot higher dilutions can be used. Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Species Rat	Dilution info 1/5000 - 1/10000	Notes For Rat and Mouse samples 1/500 dilution has only been tried. We have not tested if similarly to Human samples a lot higher dilutions can be used.
Species Human	Dilution info 1/5000 - 1/10000	Notes For Rat and Mouse samples 1/500 dilution has only been tried. We have not tested if similarly to Human samples a lot higher dilutions can be used.

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/500	Notes -
Species Rat	Dilution info 1/500	Notes -
Species Human	Dilution info 1/500	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/100 - 1/2200	Notes Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
Species Rat	Dilution info 1/100 - 1/2200	Notes Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
Species Human	Dilution info 1/100 - 1/2200	Notes Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/500	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Species Rat	Dilution info 1/500	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Species Human	Dilution info 1/500	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Associated Products

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Target data

See full target information S6K1

Function

Serine/threonine-protein kinase that acts downstream of mTOR signaling in response to growth factors and nutrients to promote cell proliferation, cell growth and cell cycle progression (PubMed:11500364, PubMed:12801526, PubMed:14673156, PubMed:15071500, PubMed:15341740, PubMed:16286006, PubMed:17052453, PubMed:17053147, PubMed:17936702, PubMed:18952604, PubMed:19085255, PubMed:19720745, PubMed:19935711, PubMed:19995915, PubMed:22017876, PubMed:23429703, PubMed:28178239). Regulates protein synthesis through phosphorylation of EIF4B, RPS6 and EEF2K, and contributes to cell survival by repressing the pro-apoptotic function of BAD (PubMed:11500364, PubMed:12801526, PubMed:14673156, PubMed:15071500, PubMed:15341740, PubMed:16286006, PubMed:17052453, PubMed:17053147, PubMed:17936702, PubMed:18952604, PubMed:19085255, PubMed:19720745, PubMed:19935711, PubMed:19995915, PubMed:22017876, PubMed:23429703, PubMed:28178239). Under conditions of nutrient depletion, the inactive form associates with the EIF3 translation initiation complex (PubMed:16286006). Upon mitogenic stimulation, phosphorylation by the mechanistic target of rapamycin complex 1 (mTORC1) leads to dissociation from the EIF3 complex and activation (PubMed:16286006). The active form then phosphorylates and activates several substrates in the pre-initiation complex, including the EIF2B complex and the cap-binding complex component EIF4B (PubMed:16286006). Also controls translation initiation by phosphorylating a negative regulator of EIF4A, PDCD4, targeting it for ubiquitination and subsequent proteolysis (PubMed:17053147). Promotes initiation of the pioneer round of protein synthesis by phosphorylating POLDIP3/SKAR (PubMed:15341740). In response to IGF1, activates translation elongation by phosphorylating EEF2 kinase (EEF2K), which leads to its inhibition and thus activation of EEF2 (PubMed:11500364). Also plays a role in feedback regulation of mTORC2 by mTORC1 by phosphorylating RICTOR, resulting in the inhibition of mTORC2 and AKT1 signaling (PubMed:19720745, PubMed:19935711, PubMed:19995915). Also involved in feedback regulation of mTORC1 and mTORC2 by phosphorylating DEPTOR (PubMed:22017876). Mediates cell survival by phosphorylating the pro-apoptotic protein BAD and suppressing its pro-apoptotic function (By similarity). Phosphorylates mitochondrial URI1 leading to dissociation of a URI1-PPP1CC complex (PubMed:17936702). The free mitochondrial PPP1CC can then dephosphorylate RPS6KB1 at Thr-412, which is proposed to be a negative feedback mechanism for the RPS6KB1 anti-apoptotic function (PubMed:17936702). Mediates TNF-alpha-induced insulin resistance by phosphorylating IRS1 at multiple serine residues, resulting in accelerated degradation of IRS1 (PubMed:18952604). In cells lacking functional TSC1-2 complex, constitutively phosphorylates and inhibits GSK3B (PubMed:17052453). May be involved in cytoskeletal rearrangement through binding to neurabin (By similarity). Phosphorylates and activates the pyrimidine biosynthesis enzyme CAD, downstream of MTOR (PubMed:23429703). Following activation by mTORC1, phosphorylates EPRS and thereby plays a key role in fatty acid uptake by adipocytes and also most probably in interferon-gamma-induced translation inhibition (PubMed:28178239).

Alternative names

STK14A, Ribosomal protein S6 kinase beta-1, S6K-beta-1, S6K1, 70 kDa ribosomal protein S6 kinase 1, Ribosomal protein S6 kinase I, Serine/threonine-protein kinase 14A, p70 ribosomal S6 kinase alpha, P70S6K1, p70-S6K 1, p70 S6 kinase alpha, p70 S6K-alpha, p70 S6KA

Recommended products

Rabbit Recombinant Monoclonal KS6B1 antibody. Suitable for IP, WB, ICC/IF, Flow Cyt (Intra), IHC-P and reacts with Mouse, Human, Rat samples. Cited in 96 publications.

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 50% Glycerol (glycerin, glycerine), 49% PBS, 0.05% BSA
Form: Liquid
Clonality: Monoclonal
Immunogen: The exact immunogen used to generate this antibody is proprietary information.
Clone number: E343
Purification technique: Affinity purification Protein A
Specificity: This antibody detects both alpha I and alpha II isoforms.
Concentration

Storage

Shipped at conditions: Blue Ice
Appropriate short-term storage conditions: +4°C
Appropriate long-term storage conditions: -20°C
Aliquoting information: Upon delivery aliquot
Storage information: Avoid freeze / thaw cycle

Notes

Patented technology
Our RabMAb^® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb^® patents.

What are the advantages of a recombinant monoclonal antibody?
This product is a recombinant monoclonal antibody, which offers several advantages including:

- High batch-to-batch consistency and reproducibility
- Improved sensitivity and specificity
- Long-term security of supply
- Animal-free batch production

For more information, read more on recombinant antibodies.

Supplementary info

This supplementary information is collated from multiple sources and compiled automatically.

Activity summary

S6K1 also known as p70S6K is a serine/threonine kinase involved in protein synthesis. The protein has a molecular weight of approximately 70 kDa. Scientists often examine the presence of S6K1 in various tissues as it is expressed ubiquitously in eukaryotic cells. This kinase plays an instrumental role in the regulation of cell growth proliferation and survival by phosphorylating ribosomal protein S6 and other targets. Researchers explore various cellular contexts where S6K1 activity is implicated due to its widespread expression and function.

Biological function summary

S6K1 functions as a downstream effector of the mammalian target of rapamycin (mTOR) complex specifically mTORC1. S6K1 interacts with several protein complexes enhancing its regulatory capacity in cellular activities. It modulates protein synthesis by phosphorylating substrates involved in translation. By controlling these processes S6K1 aids in cell size regulation and energy metabolism which are critical for maintaining cellular homeostasis and adaptation to nutrient availability.

Pathways

S6K1 plays an essential role in the mTOR signaling pathway a central regulator of cell growth and metabolism. S6K1 functions in close connection with proteins such as mTOR and Raptor within this pathway. Additionally it is involved in the insulin signaling pathway where it works with proteins like insulin receptor substrate (IRS). Both pathways highlight S6K1's role in nutrient sensing and response emphasizing its significance in energy and protein homeostasis.

Associated diseases and disorders

S6K1 is associated with conditions such as cancer and type 2 diabetes. Dysregulation of S6K1 activity can lead to abnormal cell proliferation contributing to oncogenesis in various cancer types. Specifically altered signaling through pathways involving mTOR and Akt proteins connects S6K1 to cancer progression. In type 2 diabetes S6K1 affects insulin sensitivity where overactive S6K1 signaling leads to insulin resistance. Here interactions with insulin receptor substrate proteins highlight its influence on metabolic disorders underlining its critical role in disease pathology.

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20 product images

Western blot - Anti-S6K1 antibody [E343] (ab32529)
S6K1 Western blot staining using rabbit Anti-S6K1 antibody
Lane 1: Wild-type HAP1 cell lysate (20 μg)
Lane 2: S6K1 knockout HAP1 cell lysate (20 μg)
Lane 3: MCF7 cell lysate (20 μg)
Lane 4: HEK293 cell lysate (20 μg)
Lanes 1 - 4: Merged signal (red and green). Green - ab32529 observed at 68 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - Loading Control ab8245, observed at 37 kDa.
ab32529 was shown to recognize S6K1 when S6K1 knockout samples were used, along with additional cross-reactive bands. Wild-type and S6K1 knockout samples were subjected to SDS-PAGE. ab32529 and Anti-GAPDH antibody [6C5] - Loading Control ab8245 (loading control to GAPDH) were diluted 1/5000 and 1/10000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.
All lanes: Western blot - Anti-S6K1 antibody [E343] (ab32529)
Predicted band size: 59 kDa
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-S6K1 antibody [E343] (ab32529)
Immunohistochemical analysis of rat brain tissue labeling S6K1 with ab32529 at 1/500 dilution (4.4 μg/mL). The secondary antibody used was ImmunoHistoProbe one step HRP Polymer (ready to use). Secondary antibody only control-PBS instead of the primary antibody. Antigen retrieval was heat mediated using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0). The tissue was counterstained with Hematoxylin.
Western blot - Anti-S6K1 antibody [E343] (ab32529)
S6K1 Western blot staining using rabbit Anti-S6K1 antibody
Blocking and diluting buffer used: 5% NFDM/TBST.
All lanes: Western blot - Anti-S6K1 antibody [E343] (ab32529) at 0.004 µg/mL
Lane 1: Neuro2a (Mouse neuroblastoma neuroblast) whole cell lysate at 20 µg
Lane 2: Mouse cerebellum lysate at 20 µg
Lane 3: C6 (Rat glial tumor glial cell) whole cell lysate at 20 µg
Lane 4: Rat cerebellum at 20 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution
Predicted band size: 59 kDa
Observed band size: 70 kDa
Exposure time: 3min
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-S6K1 antibody [E343] (ab32529)
Immunohistochemical analysis of mouse testis tissue labeling S6K1 with ab32529 at 1/500 dilution (4.4 μg/mL). The secondary antibody used was ImmunoHistoProbe one step HRP Polymer (ready to use). Secondary antibody only control-PBS instead of the primary antibody. Antigen retrieval was heat mediated using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0). The tissue was counterstained with Hematoxylin.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-S6K1 antibody [E343] (ab32529)
Immunohistochemical analysis of Human breast cancer tissue labeling S6K1 with ab32529 at 1/500 dilution (4.4 μg/mL). The secondary antibody used was ImmunoHistoProbe one step HRP Polymer (ready to use). Secondary antibody only control-PBS instead of the primary antibody. Antigen retrieval was heat mediated using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0). The tissue was counterstained with Hematoxylin.
Flow Cytometry (Intracellular) - Anti-S6K1 antibody [E343] (ab32529)
Intracellular Flow Cytometry analysis of C6 (Rat glial tumor glial cell) cells labelling with ab32529 (purified) at 1/2200 dilution (1μg/mL) (red). Cells were fixed with 4% paraformaldehyde . Goat anti rabbit IgG (Alexa Fluorr^® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) was used as the secondary antibody at 1/2000 dilution. Isotype control - 90% methanol . Unlabeled control - Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) / Black.
Immunoprecipitation - Anti-S6K1 antibody [E343] (ab32529)
Lane 1: Neuro2a (Mouse neuroblastoma neuroblast) whole cell lysate, 10μg
Lane 2: Neuro2a whole cell lysate 350μg and ab32529, 2μg
Lane 3: Neuro2a cell lysate, 350μg and rabbit IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730), 2μg

Purified ab32529 immunoprecipitating S6K1 in HEK293T cell lysates. Primary antibody was used at a 1/110 dilution (20 μg/ml). For western blotting, ab32529 at 1/500 and VeriBlot for IP (HRP) VeriBlot for IP Detection Reagent (HRP) ab131366 was used for detection at 1/1000 dilution.
Blocking and diluting buffer used: 5% NFDM/TBST.
All lanes: Immunoprecipitation - Anti-S6K1 antibody [E343] (ab32529)
Predicted band size: 59 kDa
Flow Cytometry (Intracellular) - Anti-S6K1 antibody [E343] (ab32529)
Intracellular Flow Cytometry analysis of 293T (Human embryonic kidney epithelial cell) cells labelling with ab32529 (purified) at 1/2200 dilution (1μg/mL) (red). Cells were fixed with 4% paraformaldehyde . Goat anti rabbit IgG (Alexa Fluorr^® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) was used as the secondary antibody at 1/2000 dilution. Isotype control - 90% methanol . Unlabeled control - Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) / Black.
Immunoprecipitation - Anti-S6K1 antibody [E343] (ab32529)
Lane 1: HEK293T (Human embryonic kidney epithelial cell) whole cell lysate, 10μg
Lane 2: HEK293T whole cell lysate, 10μg and ab32529, 2μg
Lane 3: HEK293T cell lysate, 350μg and rabbit IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) , 2μg

Purified ab32529 immunoprecipitating S6K1 in HEK293T cell lysates. Primary antibody was used at a 1/110 dilution (20 μg/ml). For western blotting, ab32529 at 1/500 and VeriBlot for IP (HRP) VeriBlot for IP Detection Reagent (HRP) ab131366 was used for detection at 1/1000 dilution.

Blocking and diluting buffer used: 5% NFDM/TBST.
All lanes: Immunoprecipitation - Anti-S6K1 antibody [E343] (ab32529)
Predicted band size: 59 kDa
Immunocytochemistry/ Immunofluorescence - Anti-S6K1 antibody [E343] (ab32529)
Immunocytochemistry/Immunofluorescence analysis of C6 cells (Rat glial tumor glial cell) labelling S6K1 with ab32529 at a dilution of 1:200, 11.1 μg/ml. Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. A 1:1000 dilution (2μg/ml) was used for the secondary antibody Goat anti rabbit IgG (Alexa Fluor® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077). The cells were co-stained with 1:200, 2.5μg/ml with Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594). Nuclei counterstained with DAPI (blue). Control: 1:1000 dilution.
Immunocytochemistry/ Immunofluorescence - Anti-S6K1 antibody [E343] (ab32529)
Immunocytochemistry/Immunofluorescence analysis of NIH/3T3 (Mouse embryonic fibroblast) labelling with ab32529 at a dilution of 1:200, 11.1 μg/ml. Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. A 1:1000 dilution (2μg/ml) was used for the secondary antibody Goat anti rabbit IgG (Alexa Fluor® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077). The cells were co-stained at 1:200 dilution, 2.5μg/ml with Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594). Nuclei counterstained with DAPI (blue). Control: 1:1000 dilution.
Immunocytochemistry/ Immunofluorescence - Anti-S6K1 antibody [E343] (ab32529)
Immunocytochemistry/Immunofluorescence analysis of MCF 7 (Human breast adenocarcinoma epithelial cell) labeling S6K1 with ab32529 at a dilution of 1:200, 11.1 ug/ml. Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. A dilution of 1/1000 (2μg/ml) was used for the secondary antibodyGoat anti rabbit IgG (Alexa Fluor® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077). The cells were co-stained at 1:200 dilution, 2.5μg/ml with Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) . Nuclei counterstained with DAPI (blue). Control: 1:1000 dilution.
Immunocytochemistry/ Immunofluorescence - Anti-S6K1 antibody [E343] (ab32529)
Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) labeling S6K1 with ab32529 at a dilution of 1/500. Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% tritonX-100. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 at 1/1000 was used as the secondary antibody. Nuclei were counterstained with DAPI (blue). Confocal image showing cytoplamic staining on HeLa cell line.
Flow Cytometry (Intracellular) - Anti-S6K1 antibody [E343] (ab32529)
Overlay histogram showing HeLa cells stained with ab32529 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32529, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.
Western blot - Anti-S6K1 antibody [E343] (ab32529)
S6K1 Western blot staining of 293T cell lysate using rabbit Anti-S6K1 antibody
All lanes: Western blot - Anti-S6K1 antibody [E343] (ab32529) at 1/10000 dilution
All lanes: 293T cell lysate
Predicted band size: 59 kDa
Observed band size: 70 kDa
Flow Cytometry (Intracellular) - Anti-S6K1 antibody [E343] (ab32529)
Intracellular Flow Cytometry analysis of Neuro-2a (Mouse neuroblastoma neuroblast) cells labelling with ab32529 (purified) at 1/2200 dilution (1μg/mL) (red). Cells were fixed with 4% paraformaldehyde . Goat anti rabbit IgG (Alexa Fluorr^® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) was used as the secondary antibody at 1/2000 dilution. Isotype control - 90% methanol . Unlabeled control - Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) / Black.
Western blot - Anti-S6K1 antibody [E343] (ab32529)
S6K1 Western blot staining using rabbit Anti-S6K1 antibody
Western blot: Rabbit Monoclonal[E343] to S6K1 ab32529 staining at 1/10000 dilution, shown in green; Mouse anti GAPDH (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 59 kDa in Wild-type U-87 MG cell lysates with no signal observed at this size in RPS6KB1 knockout U-87 MG cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.
All lanes: Western blot - Anti-S6K1 antibody [E343] (ab32529) at 1/10000 dilution
Lane 1: Wild-type U-87 MG at 20 µg
Lane 2: RPS6KB1 knockout U-87 MG at 20 µg
Lane 3: MCF7 at 20 µg
Lane 4: HEK-293 at 20 µg
Secondary
All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 59 kDa
Observed band size: 59 kDa
Western blot - Anti-S6K1 antibody [E343] (ab32529)
S6K1 Western blot staining using rabbit Anti-S6K1 antibody
Blocking and diluting buffer and concentration: 5% NFDM/TBST.
The identity of the higher MW band at approximately 150 kDa (in lane 2) is unknown.
In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) staining at 1/200000 dilution.
In Western blot, Anti-RPS6KB1 antibody [E343] (ab32529) staining at 1/1000 dilution.
All lanes: Western blot - Anti-S6K1 (phospho T389) antibody [EPR24766-9] (Anti-S6K1 (phospho T389) antibody [EPR24766-9] ab323272) at 1/1000 dilution
Lane 1: Untreated HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 50 µg
Lane 2: Hela treated with 100nM Calycin A for 30min whole cell lysate at 50 µg
Lane 3: Hela treated with 100nM Calycin A for 30min whole cell lysate (alkaline phosphatase treated membrane) at 50 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution
Observed band size: 60-80 kDa, 36 kDa
Exposure time: 37s
Western blot - Anti-S6K1 antibody [E343] (ab32529)
S6K1 Western blot staining using rabbit Anti-S6K1 antibody
Blocking and diluting buffer and concentration: 5% NFDM/TBST.
Performed under reducing conditions.
In Western blot, Anti-S6K1 (phospho T389) antibody [EPR24766-9] ab323272 was shown to bind specifically to RPS6KB1. Target of interest was observed at 60–80kDa in wild-type HAP1 cell lysates (lane 2) with no signal observed at this size in RPS6KB1 knockout cell line (lane 4).
The identity of the higher MW band at approximately 150 kDa (in lanes 2/4) is unknown.
In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) staining at 1/200000 dilution.
In Western blot, Anti-RPS6KB1 antibody [E343] (ab32529) staining at 1/1000 dilution.
All lanes: Western blot - Anti-S6K1 (phospho T389) antibody [EPR24766-9] (Anti-S6K1 (phospho T389) antibody [EPR24766-9] ab323272) at 1/1000 dilution
Lane 1: HAP1(HOWT01) (human chronic myelogenous leukemia near-haploid cell) whole cell lysate at 50 µg
Lane 2: Parental HAP1(HOWT01) treated with 100nM Calycin A for 30min whole cell lysate at 50 µg
Lane 3: Untreated RPS6KB1 knockout HAP1 whole cell lysate at 50 µg
Lane 4: RPS6KB1 knockout HAP1 treated with 100nM Calycin A whole cell lysate at 50 µg
Lane 5: Untreated parental HAP1(HOWT01) (human chronic myelogenous leukemia near-haploid cell) whole cell lysate (alkaline phosphatase treated membrane) at 50 µg
Lane 6: Parental HAP1(HOWT01) treated with 100nM Calyculin A for 30min whole cell lysate (alkaline phosphatase treated membrane) at 50 µg
Lane 7: Untreated RPS6KB1 knockout HAP1 whole cell lysate (alkaline phosphatase treated membrane) at 50 µg
Lane 8: RPS6KB1 knockout HAP1 treated with 100nM Calyculin A whole cell lysate (alkaline phosphatase treated membrane) at 50 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution
Performed under reducing conditions.
Observed band size: 60-80 kDa, 36 kDa
Exposure time: 180s
Image collected and cropped by CiteAb under a CC-BY license from the publication
Western blot - Anti-S6K1 antibody [E343] (ab32529)
S6K1 western blot using anti-S6K1 antibody [E343] ab32529. Publication image and figure legend from Liu, S. C., Hsu, T., et al., 2018, Nat Commun, PubMed 30504771.

ab32529 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab32529 please see the product overview.
Characterization of LIF mutant clones. a Sequence analysis of the LIF gene. Genomic DNA was extracted from parental NPC BM1 cells with wild-type LIF or established clones either with mutations in the signal peptide region of LIF (cLIF clone) or loss of the initiating codon in one allele (LIF+/− clone). The initiating codon within the spacer is indicated in red. Mutated nucleotides are marked in blue. b Assessment of LIF protein expression via western blot using GAPDH as a loading control. c Assessment of secreted LIF using a bead-based cytokine assay. Supernatants were harvested 2 days post culture. Data are presented as means ± SD of triplicate experiments. **p < 0.01, two-tailed, paired t test. d Immunofluorescent detection of LIF (green) in WT, cLIF and LIF+/− cancer cells. Blue, nuclear staining. Scale bars, 10 μm. e Comparison of morphological changes (DIC images) in WT, cLIF, and LIF+/− cancer cells. Scale bars, 10 μm. f Live images of LIF uptake in cancer cells expressing LifeAct-RFP. Recombinant LIF proteins were pre-labeled with ATTO 488 green fluorescent dye. Images were captured 40 min post-LIF addition. g Time-course analysis of LIFR desensitization and p70S6K1 activation in LIF (30 ng/ml)-stimulated cells using GAPDH as a loading control