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

Lane 1 : LNCaP (Human prostate carcinoma epithelial cell) whole cell lysates
Lane 2 : LNCaP (Human prostate carcinoma epithelial cell) treated with 0.1 uM Calyculin A for 30 minutes whole cell lysates
Lane 3 : A549 (Human lung carcinoma epithelial cell) whole cell lysates
Lane 4 : MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysates
Lane 5 : HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysates
Lane 6 : HUVEC (Human umbilical vein endothelial cell) whole cell lysates
Lane 7 : C2C12 (Mouse myoblasts myoblast) whole cell lysates
Lane 8 : Mouse brain lysates
Lane 9 : Rat heart lysates

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 56 kDa
Observed band size: 56 kDa


Exposure time: 50 seconds

The basal expression level of AKT1 (phospho S473) varies in different cell lines reported by PMID: 19372546. But to detect clear signal, treatment is strongly recommended when using this antibody.

Blocking and Diluting buffer: 5% NFDM/TBST

Immunofluorescent 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.

Dot blot analysis of AKT3 (phospho S472) labeled with ab192623 at 1/1000 dilution.

Lane 1: AKT3 (phospho S472) phospho peptide;

Lane 2: AKT3 non-phospho peptide;

Lane 3: AKT1 (phospho S473) phospho peptide;

Lane 4: AKT2 (phospho S474) phospho peptide.

Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated (ab97051) at 1/100000 dilution was used as secondary antibody.

Blocking and diluting buffer: 5% NFDM/TBST.

Exposure time: 3 minutes.

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

Lane 1 : Untreated MCF7 (Human breast adenocarcinoma cell line) whole cell lysate
Lane 2 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate treated with 100ng/ml IGF-1 for 15 minutes

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 56 kDa
Observed band size: 56 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Phosphorylation of AKT at S473 can be induced by IGF-1 treatment according to the literature (PMID: 23638184).

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

Lane 1 : Untreated PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 2 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate treated with 100ng/ml PDGF for 60 minutes
Lane 3 : Untreated NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate
Lane 4 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate treated with 100ng/ml PDGF for 60 minutes

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 56 kDa
Observed band size: 56 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1 and 2: 15 seconds; Lane 3 and 4: 3 minutes.

Phosphorylation of AKT can be induced by PDGF treatment according to the literature (PMID: 10984605 and 7774014).

AKT3 (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.