Anti-S6K1 antibody [E343]

• ICC/IF

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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. 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.

• IHC-P

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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 ab93684 (Tris/EDTA buffer, pH 9.0). The tissue was counterstained with Hematoxylin.

• ICC/IF

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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, ab150077). The cells were co-stained at 1 : 200 dilution, 2.5μg/ml with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) . Nuclei counterstained with DAPI (blue). Control : 1 : 1000 dilution.

• Flow Cyt (Intra)

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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) (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.

• Flow Cyt (Intra)

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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, ab150077) was used as the secondary antibody at 1/2000 dilution. Isotype control - 90% methanol . Unlabeled control - Rabbit monoclonal IgG (ab172730) / Black.

• IP

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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 (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) 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

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• IHC-P

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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 ab93684 (Tris/EDTA buffer, pH 9.0). The tissue was counterstained with Hematoxylin.

• IHC-P

Unknown

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 ab93684 (Tris/EDTA buffer, pH 9.0). The tissue was counterstained with Hematoxylin.

• Flow Cyt (Intra)

Unknown

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, ab150077) was used as the secondary antibody at 1/2000 dilution. Isotype control - 90% methanol . Unlabeled control - Rabbit monoclonal IgG (ab172730) / Black.

• ICC/IF

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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, ab150077). The cells were co-stained with 1 : 200, 2.5μg/ml with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594). Nuclei counterstained with DAPI (blue). Control : 1 : 1000 dilution.

• Flow Cyt (Intra)

Lab

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, ab150077) was used as the secondary antibody at 1/2000 dilution. Isotype control - 90% methanol . Unlabeled control - Rabbit monoclonal IgG (ab172730) / Black.

• ICC/IF

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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, ab150077). The cells were co-stained at 1 : 200 dilution, 2.5μg/ml with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594). Nuclei counterstained with DAPI (blue). Control : 1 : 1000 dilution.

• IP

Unknown

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 (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) 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

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• WB

Supplier Data

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

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 (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] (<a href='/en-us/products/primary-antibodies/s6k1-phospho-t389-antibody-epr24766-9-ab323272'>ab323272</a>) 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) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 60-80 kDa,36 kDa

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Exposure time: 37s

• WB

Lab

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

Western blot : Rabbit Monoclonal[E343] to S6K1 ab32529 staining at 1/10000 dilution, shown in green; Mouse anti GAPDH (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 (ab326023). 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:

Western blot - Human RPS6KB1 knockout U-87 MG cell line (<a href='/en-us/products/cell-lines/human-rps6kb1-knockout-u-87-mg-cell-line-ab326023'>ab326023</a>) 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

Predicted band size: 59 kDa

Observed band size: 59 kDa

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• WB

Lab

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

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) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 59 kDa

Observed band size: 70 kDa

false

Exposure time: 3min

• WB

Lab

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

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-RPS6KB1 antibody [E343] - Total protein control (ab32529) staining at 1/1000 dilution.

The identity of the bands at approximately 150 kDa (in lane 2) and at approximately 30 kDa (in lanes 1-2) are unknown.

All lanes:

Western blot - Anti-S6K1 (phospho T389) antibody [EPR24766-9] (<a href='/en-us/products/primary-antibodies/s6k1-phospho-t389-antibody-epr24766-9-ab323272'>ab323272</a>) at 1/1000 dilution

Lane 1:

Untreated NIH/3T3 (mouse embryonic fibroblast) starved overnight, whole cell lysate at 20 µg

Lane 2:

NIH/3T3 starved overnight, then treated with 100nM Calyculin A for 30min whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/200000 dilution

Observed band size: 60-80 kDa

false

Exposure time: 180s

• WB

Unknown

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

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

false

• WB

CiteAb

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

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