Western blotting analysis using phospho-PRAS40 (T246) antibody (Cat#4666). 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 (Cat#4666, 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).
Immunohistochemistry was performed on paraffin-embedded human hepatocarcinoma using phospho-PRAS40 (T246) antibody (Cat#4666, 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.
Immunocytochemical staining of HeLa cells with Phospho-PRAS40 (T246) antibody (Cat#4666, 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. 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 Phospho-PRAS40 (T246) expression in HeLa cells using Phospho-PRAS40 (T246) antibody (Cat#4666, 1:2,000). Fixative solution: 4% Paraformaldehyde in 1x PBS; Permeabilization solution: 100% Methanol, chill on -20℃ before use. Green, isotype control; red, Phospho-PRAS40 (T246).
