PI3K/AKT signalling pathway panel (ab283852)

All lanes: Anti-AKT1 + AKT2 + AKT3 (phospho S472 + S473 + S474) antibody [EPR18853] (ab192623) at 1/1000 dilution.

Lane 1:  LNCaP (Human prostate carcinoma epithelial cell) whole cell lysates, 20 µg

Lane 2:  LNCaP (Human prostate carcinoma epithelial cell) treated with 0.1 uM Calyculin A for 30 minutes whole cell lysates, 20 µg

Lane 3:  A549 (Human lung carcinoma epithelial cell) whole cell lysates, 20 µg

Lane 4: MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysates, 20 µg

Lane 5: HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysates, 20 µg

Lane 6: HUVEC (Human umbilical vein endothelial cell) whole cell lysates, 20 µg

Lane 7: C2C12 (Mouse myoblasts myoblast) whole cell lysates, 20 µg

Lane 8: Mouse brain lysates, 20 µg

Lane 9: Rat heart lysates, 20 µg

Secondary (all lanes): Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted MW: 56 kDa.

Observed MW: 56 kDa.

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: 50 seconds.

KT3 (phospho S472) was immunoprecipitated from 0.35 mg of NIH/3T3 (Mouse embryonic fibroblast cell line) treated with 50ng/ml PDGF for 40min whole cell lysate with ab192623 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab192623 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Lane 1: NIH/3T3 treated with 50ng/ml PDGF for 40min whole cell lysate, 10μg (Input).

Lane 2: ab192623 IP in NIH/3T3 treated with 50ng/ml PDGF for 40min whole cell lysate.

Lane 3: Rabbit IgG,monoclonal [EPR25A]-Isotype Control (ab172730) instead of ab192623 in NIH/3T3 treated with 50ng/ml PDGF for 40min whole cell lysate.

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

Exposure time: 5 seconds.

Lanes 1 - 3: Merged signal (red and green). Green - ab134903 observed at 289 kDa. Red - loading control, ab130007, observed at 130 kDa.

Lane 1: Wild-type HEK293T whole cell lysate, 20 µg

Lane 2: MTOR knockout HEK293T whole cell lysate, 20 µg

Lane 3: K562 whole cell lysate, 20 µg

ab134903 was shown to specifically react with mTOR in wild-type HEK293T cells as signal was lost in MTOR knockout cells. Wild-type and MTOR knockout samples were subjected to SDS-PAGE. Ab134903 and ab130007 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/10000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes: Anti-PI 3 Kinase p85 alpha (phospho Y607) antibody (ab182651) at 1/500 dilution.

Lane 1: Insulin treated HeLa cell lysates

Lane 2: HeLa cell lysates

Secondary (all lanes): HRP-conjugate secondary antibody.

Predicted MW: 83 kDa.

Blocking/Dilution buffer: 5% non-fat milk.

Lanes 1 - 3: Merged signal (red and green). Green - ab191606 observed at 90 kDa. Red - loading control, ab9484, observed at 37 kDa.

Lane 1: Wild type HAP1 whole cell lysate, 20 µg
Lane 2: PIK3R1 knockout HAP1 whole cell lysate, 20 µg
Lane 3: Jurkat whole cell lysate, 20 µg

ab191606 was shown to specifically react with PIK3R1 when PIK3R1 knockout samples were used. Wild-type and PIK3R1 knockout samples were subjected to SDS-PAGE. Ab191606 and ab9484 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes: Anti-AKT1 + AKT2 + AKT3 antibody [EPR16798] (ab179463) at 1/1000 dilution.

Lane 1: MCF7 (Human breast adenocarcinoma cell line) whole cell lysates, 20 µg

Lane 2: HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysates, 20 µg

Lane 3: Hep G2 (Human liver hepatocellular carcinoma) whole cell lysates, 20 µg

Secondary (all lanes):  Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution.

Predicted MW: 56 kDa.

Observed MW: 56 kDa.

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: 70 seconds.

Immunocytochemical analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embryonic fibroblast cell line) cells, untreated or treated with PDGF (100 ng/ml) for 1 hour, labeling AKT3 (phospho S472) + AKT2 (phospho S474) + AKT1 (phospho S473) with ab192623 at 1/100 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

The signal increased after treatment with PDGF (100 ng/ml) for 1 hour on NIH/3T3 cells.

The nuclear counter stain is DAPI (blue).

Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)) at 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HepG2 (Human liver hepatocellular carcinoma cell line) cells labeling PI3K p85 with ab191606 at 1/250 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on HepG2 cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody - Loading Control (ab7291)  at 1/1000 dilution and Goat Anti-Mouse IgG (AlexaFluor^®594) preadsorbed (ab150120)  at 1/1000 dilution (red).

The negative controls are as follows:
-ve control 1: ab191606 at 1/500 dilution followed by ab150120  at 1/1000 dilution.
-ve control 2: ab7291  at 1/1000 dilution followed by ab150077 at 1/1000 dilution.

Immunocytochemical analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized K562 (Human chronic myelogenous leukemia cells from bone marrow) cells labeling AKT1 + AKT2 + AKT3 with ab179463 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/400 dilution (green). Cytoplasm and nuclear staining on K562 cell line is observed. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).

The negative controls are as follows:
1. ab179463 at 1/100 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/400 dilution.

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling AKT1 + AKT2 + AKT3 with ab179463 at 1/250 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Cytoplasm and nucleus staining on Human renal cortex is observed. Counterstained with Hematoxylin.

Negative control: Using PBS instead of primary ab, secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Rat cerebral cortex tissue labeling AKT1 + AKT2 + AKT3 with ab179463 at 1/250 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Cytoplasm and nucleus staining on Rat cerebral cortex is observed. Counter stained with Hematoxylin.

Negative control: Using PBS instead of primary ab, secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Flow cytometric analysis of 4% paraformaldehyde-fixed NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling PI3K p85 with ab191606 at 1/150 dilution (red) compared with a Rabbit IgG,monoclonal -  Isotype control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti Rabbit IgG (Alexa Fluor^® 488) at 1/500 dilution was used as the secondary antibody.

Flow cytometric analysis of 2% paraformaldehyde-fixed A549 (Human lung carcinoma) cells labeling AKT1 + AKT2 + AKT3 with ab179463 at 1/330 dilution (red) compared with a rabbit monoclonal IgG isotype control (black) and a unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/150 dilution was used as the secondary antibody.