Analysis of PKCα Kinase Activity
Human MDMs were starved for 2 hours before stimulation with M-CSF (0–60 minutes), then washed with cold PBS and removed from the plates. The cells were resuspended in lysis buffer (20 mM Tris, 5 mM MgCl2, 1 mM EGTA, 20 mM β-glycerol phosphate, 1 mM PMSF 2 µg/ml aprotinin and 2 µg/ml leupeptin, pH 7.5), sonicated and centrifuged to obtain whole cell lysates, which were then immunoprecipitated with anti-PKCα antibody and protein G-agarose (Invitrogen). Immune complexes were washed twice, and PKCα activity was analyzed by non-radioactive Peptag assay kit (Promega). Briefly, kinase buffer, activator, peptide protection solution and Peptag peptide were incubated with the immune complexes at 30°C for 30 minutes. Reactions were stopped by boiling for 10 minutes and samples were separated on 0.8% agarose gel. Phosphorylated peptide migrated toward the anode (+), while non-phosphorylated peptide migrated toward the cathode (−). Fluorescein-tagged peptides were visualized by UV light.