Smoke stimulates phosphorylation and activation of TACE.
(A) NCIH292 cells were incubated with SFM, smoke containing SFM (SMK), EGF (10 ng/ml)-containing SFM, or LTA (50 µg/ml)-containing SFM for 10 min. Cell lysates were immunoprecipitated (IP) with anti-TACE antibody, immunoblotted (IB) with anti-phosphoserine, antiphosphothreonine, anti-phosphotyrosine and anti-TACE antibodies, and visualized by chemiluminescence. P and M indicate the Pro- and Mature forms of TACE, respectively. (B) TACE activity was measured using the fluorescent InnoZyme™ TACE activity Kit. NCIH292 cells were incubated with SFM, smoke containing SFM (SMK) for 10 min then harvested. Total cell lysates were prepared and TACE activity was measured according to the manufacturer's recommended protocol. RFU is reported per mg protein. To assess the relevance of phosphorylation on TACE catalytic activity, NCIH292 cells were incubated with phosphatase at the indicate concentrations for 2 h prior to stimulation with smoke (SMK) for 10 min. Double asterisks indicate significantly different from SFM (p<0.01).