VALIDATION IMAGES
Lane 1: NIH/3T3 treated with PDGF; Lane 2: NIH/3T3 untreatedlysates probed with Akt1(Ser473) (12A1) Monoclonal Antibody (bsm-52130R) at 1:1000 overnight at 4˚C. Followed by a conjugated secondary antibody. The bottom panel is antibody staining using an unphosphorylated AKT1 antibody.
3T3 cells were stained with Akt1(Ser473) (12A1) Monoclonal Antibody (bsm-52130R) at [1:200] incubated overnight at 4C, followed by secondary antibody incubation, DAPI staining of the nuclei and detection.
Paraformaldehyde-fixed, paraffin embedded mouse lung section; Antigen retrieval by boiling in sodium citrate buffer (pH6) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 30 minutes; Blocking buffer (normal goat serum) at 37°C for 20min; Antibody incubation with Akt1(Ser473) (12A1) Monoclonal Antibody (bsm-52130R) at 1:400 overnight at 4°C, followed by a conjugated secondary and DAB staining.
MCF7 cells (black) were fixed with 4% PFA for 10min at room temperature, permeabilized with 90% ice-cold methanol for 20 min at -20℃, and incubated in 5% BSA blocking buffer for 30 min at room temperature. Cells were then stained with Akt1 (Ser473) (12A1) Monoclonal Antibody (bsm-52130R) at 1:50 dilution in blocking buffer and incubated for 30 min at room temperature, washed twice with 2%BSA in PBS, followed by secondary antibody(blue) incubation for 40 min at room temperature. Acquisitions of 20,000 events were performed. Cells stained with primary antibody (green), and isotype control (orange).
Paraformaldehyde-fixed, paraffin embedded Mouse testis; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with Akt1(Ser473) (12A1) Monoclonal Antibody, Unconjugated (bsm-52130R) at 1:200 overnight at 4°C, DAB staining.
U-937 cells were fixed with 4% PFA for 10min at room temperature,permeabilized with 90% ice-cold methanol for 20 min at room temperature, and incubated in 5% BSA blocking buffer for 30 min at room temperature. Cells were then stained with Akt1(Ser473) (12A1) Monoclonal Antibody(bsm-52130R)at 1:50 dilution in blocking buffer and incubated for 30 min at room temperature, washed twice with 2%BSA in PBS, followed by secondary antibody incubation for 40 min at room temperature. Acquisitions of 20,000 events were performed. Cells stained with primary antibody (green), and isotype control (orange).
Paraformaldehyde-fixed, paraffin embedded Mouse brain; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with Akt1(Ser473) (12A1) Monoclonal Antibody, Unconjugated (bsm-52130R) at 1:200 overnight at 4°C, DAB staining.
Blocking buffer: 5% NFDM/TBST Primary ab dilution: 1:2000 Primary ab incubation condition: 2 hours at room temperature Secondary ab: Goat Anti-Rabbit IgG H&L (HRP) Lysate: (-): Jurkat, (+): Jurkat+ Calyculin A (100nM, 30min) Protein loading quantity: 20 μg Exposure time: 30 s Predicted MW: 56 kDa Observed MW: 56, 60 kDa
Blocking buffer: 5% NFDM/TBST Primary ab dilution: 1:2000 Primary ab incubation condition: 2 hours at room temperature Secondary ab: Goat Anti-Rabbit IgG H&L (HRP) Lysate:(-): MCF-7, (+): MCF-7+ Calyculin A (100nM, 30min) Protein loading quantity: 20 μg Exposure time: 30 s Predicted MW: 56 kDa Observed MW: 56, 60 kDa
Blocking buffer: 5% NFDM/TBST Primary ab dilution: 1:2000 Primary ab incubation condition: 2 hours at room temperature Secondary ab: Goat Anti-Rabbit IgG H&L (HRP) Lysate:(-): HeLa, (+): HeLa+ Calyculin A (100nM, 30min) Protein loading quantity: 20 μg Exposure time: 30 s Predicted MW: 56 kDa Observed MW: 56, 60 kDa
Blocking buffer: 5% NFDM/TBST Primary ab dilution: 1:2000 Primary ab incubation condition: 2 hours at room temperature Secondary ab: Goat Anti-Rabbit IgG H&L (HRP) Lysate:(-): NIH/3T3, (+): NIH/3T3+ Calyculin A (100ng/ml, 60min) Protein loading quantity: 20 μg Exposure time: 30 s Predicted MW: 56 kDa Observed MW: 56, 60 kDa
Lane 1: Human Jurkat cell lysates; Lane 2: Human MOLT4 cell lysates probed with Phospho-Akt1(Ser473) Monoclonal Antibody, Unconjugated (bsm-52130R) at 1:300 dilution and 4°C overnight incubation. Followed by conjugated secondary antibody incubation at 1:20000 for 60 min at 37˚C.
Lane 1: Human HeLa cell lysates; Lane 2: Human Hela treated with 100nM Calyculin A for 30min; Lane 3: Human MCF-7 cell lysates; Lane 4: Human MCF-7 treated with 100nM Calyculin A for 30min probed with Phospho-Akt1 (Ser473) Monoclonal Antibody, Unconjugated (bsm-52130R) at 1:1000 dilution and 4°C overnight incubation. Followed by conjugated secondary antibody incubation at room temperature for 60 min.