Western blotting analysis using Phospho-PKC alpha (Ser657) antibody (Cat#69411). Total cell lysates (30 μg) from various cell lines were loaded and separated by SDS-PAGE. The blot was incubated with Phospho-PKC alpha (Ser657) antibody (Cat#69411, 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 Phospho-PKC alpha (Ser657) antibody (Cat#69411). Phospho-PKC alpha (Ser657) expression in wild type (WT) and Phospho-PKC alpha (Ser657) shRNA knockdown (KD) HT-1080 cells with 30 μg of total cell lysates. β-Tubulin serves as a loading control. The blot was incubated with Phospho-PKC alpha (Ser657) antibody (Cat#69411, 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 Phospho-PKC alpha (Ser657) antibody (Cat#69411, 1:1,000). Nuclei were stained blue with DAPI; Phospho-PKC alpha (Ser657) 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: 100% Methanol. Scale bar: 20 μm.
Flow cytometric analysis of Phospho-PKC alpha (Ser657) expression in HT-1080 cells using Phospho-PKC alpha (Ser657) antibody (Cat#69411, 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 (Ser657).
Validation of PKC alpha knockdown using flow cytometry. Wild-type(WT, Blue) and knockdown(KD, Green) HT1080 cells were stained with Phospho-PKC alpha (Ser657) antibody (Cat#69411, 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.
