Western blotting analysis using PRAS40 antibody (Cat#65171). Total cell lysates (30 μg) from various cell lines were loaded and separated by SDS-PAGE. The blot was incubated with pPRAS40 antibody (Cat#65171, 1:5,000) and HRP-conjugated goat anti-rabbit secondary antibody (Cat#201, 1:50,000) respectively. Image was developed using FeQ™ ECL Substrate Kit (Cat#226).
Western blotting analysis using PRAS40 antibody (Cat#65171). PRAS40 expression in wild type (WT) and PRAS40 shRNA knockdown (KD) HeLa cells with 30 μg of total cell lysates. Hsp90 α serves as a loading control. The blot was incubated with PRAS40 antibody (Cat#65171, 1:5,000) and HRP-conjugated goat anti-rabbit secondary antibody (Cat#201, 1:20,000) respectively. Image was developed using FeQ™ ECL Substrate Kit (Cat#226).
Immunocytochemical staining of HT-1080 cells with PRAS40 antibody (Cat#65171, 1:1,000). Nuclei were stained blue with DAPI; PRAS40 was stained magenta with Alexa Fluor® 647. Images were taken using Leica stellaris 5. Protein abundance based on laser Intensity and smart gain: Medium. Fixative solution: 4% Paraformaldehyde in 1x PBS; Permeabilization solution: 0.5% Triton X-100 in 1x PBS. Scale bar: 20 μm.
Flow cytometric analysis of PRAS40 expression in HT-1080 cells using PRAS40 antibody (Cat#65171, 1:2,000). Fixative solution: 4% Paraformaldehyde in 1x PBS; Permeabilization solution: 100% Methanol, chill on -20℃ before use. Green, isotype control; red, PRAS40.
Validation of PRAS40 knockdown using flow cytometry. Wild-type(WT, Blue) and knockdown(KD, Green) HeLa cells were stained with PRAS40 antibody (Cat#65171, 1:2,000) and analyzed using BD flow cytometer. Fixative solution: 4% Paraformaldehyde in 1x PBS; Permeabilization solution: 100% Methanol, chill on -20℃ before use.
