Western blotting analysis using Phospho-raf1 (S621) antibody (Cat#61511). Total cell lysates (30 μg) from various cell lines were loaded and separated by SDS-PAGE. The blot was incubated with Phospho-raf1 (S621) antibody (Cat#61511, 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).
Western blotting analysis using Phospho-raf1 (S621) antibody (Cat#61511). Phospho-raf1 (S621) expression in wild type (WT) and phospho-raf1 (S621) 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-raf1 (S621) antibody (Cat#61511, 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 sigmoid colon carcinoma using phospho-raf1 (S621) antibody (Cat#61511, 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-Raf1 (S621) antibody (Cat#61511, 1:1,000). Nuclei were stained blue with DAPI; Phospho-Raf1 (S621) was stained magenta with Alexa Fluor® 647. Images were taken using Leica stellaris 5. Protein abundance based on laser Intensity and smart gain: High. 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-Raf1 (S621) expression in HeLa cells using Phospho-Raf1 (S621) antibody (Cat#61511, 1:2,000). Fixative solution: 4% Paraformaldehyde in 1x PBS; Permeabilization solution: 100% Methanol, chill on -20℃ before use. Green, isotype control; red, Phospho-Raf1 (S621).
