Immunofluorescent analysis of Phospho-c-Raf (Ser621) in HepG2 cells (treated with 50mM Calyculin A for 30min) using a Phospho-c-Raf (Ser621) monoclonal antibody (Product # MA5-33196) at a dilution of 1:100. The cells were fixed in 4% formaldehyde, permeabilized using 0.2% Triton X-100 and blocked in 10% normal Goat Serum. The cells were then incubated with the antibody overnight at 4°C. The secondary antibody was Alexa Fluor 488-congugated Goat Anti-Rabbit IgG (H+L). Cells were counter-stained with DAPI.