Flow cytometric analysis of Phospho-PRAS40 (T246) expression in HeLa cells using Phospho-PRAS40 (T246) antibody (R021439, 1:2,000). Green, isotype control; red, Phospho-PRAS40 (T246).

Validation of PRAS40 knockdown using flow cytometry. Wild-type(WT, Blue) and knockdown(KD, Green) HeLa cells were stained with Phospho-PRAS40 (T246) antibody (R021439, 1:2,000) and analyzed using BD flow cytometer.

Immunocytochemical staining of HeLa cells with Phospho-PRAS40 (T246) antibody (R021439, 1:1,000). Nuclei were stained blue with DAPI; Phospho-PRAS40 (T246) was stained magenta with Alexa Fluor® 647. Images were taken using Leica stellaris 5. Protein abundance based on laser Intensity and smart gain: Low. Scale bar: 20 μm.

Immunohistochemistry was performed on paraffin-embedded human hepatocarcinoma using phospho-PRAS40 (T246) antibody (R021439, 1:200). Antigen retrieval was done in sodium citrate buffer (pH 6.0). DAB was used for detection, with hematoxylin counterstaining. Images were acquired using a Nikon Ci-L Plus microscope (40× objective). Scale bar: 25 μm.

Western blotting analysis using Phospho-PRAS40 (T246) antibody (R021439). Total cell lysates (30 μg) from various cell lines were loaded and separated by SDS-PAGE. The blot was incubated with Phospho-PRAS40 (T246) antibody (R021439, 1:5,000) and HRP-conjugated goat anti-rabbit secondary antibody (1:20,000) respectively. Image was developed using ECL Substrate Kit.

Western blotting analysis using Phospho-PRAS40 (T246) antibody (R021439). Phospho-PRAS40 (T246) expression in wild type (WT) and Phospho-PRAS40 (T246) shRNA knockdown (KD) HeLa cells with 30 μg of total cell lysates. Hsp90 α serves as a loading control. The blot was incubated with Phospho-PRAS40 (T246) antibody (R021439, 1:5,000) and HRP-conjugated goat anti-rabbit secondary antibody (1:20,000) respectively. Image was developed using ECL Substrate Kit.