PLCg assay

Vehicle and p43 treated THP1 cells were harvested at 1300rpm and washed 2 times with ice cold 1X PBS. Cells were lysed by addition of lysis buffer(50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2 mM EGTA, 10 mM NaF, 12 mM b-glycerophosphate, 1 mM Na₃VO_4, 10% glycerol, 1% Nonidet P-40, 5ug/ml aprotinin, 1 mM PMSF, 1 mM DTT) and incubated for 20min at 4 degree. Equal amounts(500 ug) of the proteins extracted from each treatment condition were mixed with 5ug of anti-PLCg antibody(Santa Cruz) and incubated for 2hrs. 2 hours later, 50ul of protein G conjugated agarose were added and further incubated for 2hrs. Immunoprecipitates were washed 3 times with lysis buffer and precipitated PLCg was resolved with 7.5% SDS-PAGE. PLCg and phospho-PLCg was detectedwith anti-PLCg antibody and PY20(BD Transduction Laboratories).