Western blotting analysis using Phospho-PKC alpha (T497) antibody (Cat#61850). Total cell lysates (30 μg) from various cell lines were loaded and separated by SDS-PAGE. The blot was incubated with Phospho-PKC alpha (T497) antibody (Cat#61850, 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-PKC alpha (T497) antibody (Cat#61850). Phospho-PKC alpha (T497) expression in wild type (WT) and Phospho-PKC alpha (T497) shRNA knockdown (KD) HT-1080 cells with 30 μg of total cell lysates. Hsp90 α serves as a loading control. The blot was incubated with Phospho-PKC alpha (T497) antibody (Cat#61850, 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 lung adenocarcinoma using phospho-PKC alpha (T497) antibody (Cat#61850, 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.
Immunohistochemistry was performed on paraffin-embedded human melanoma using phospho-PKC alpha (T497) antibody (Cat#61850, 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 C2C12 cells with Phospho-PKC alpha (T497) antibody (Cat#61850, 1:1,000). Nuclei were stained blue with DAPI; Phospho-PKC alpha (T497) 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.
Immunocytochemical staining of HT-1080 cells using Phospho-PKC alpha (T497) antibody (Cat#61850, 1:1,000), Top panel: wild-type (WT); Bottom panal: Phospho-PKC alpha (T497) shRNA knockdown (KD). Nuclei were stained blue with DAPI; Phospho-PKC alpha (T497) was stained magenta with Alexa Fluor® 647. 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-PKC alpha (T497) expression in C2C12 cells using Phospho-PKC alpha (T497) antibody (Cat#61850, 1:2,000). Fixative solution: 4% Paraformaldehyde in 1x PBS; Permeabilization solution: 100% Methanol, chill on -20℃ before use. Green, isotype control; red, Phospho-PKC alpha (T497).
Validation of PKC alpha knockdown using flow cytometry. Wild-type(WT, Blue) and knockdown(KD, Green) HT1080 cells were stained with Phospho-PKC alpha (T497) antibody (Cat#61850, 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.
