Fig. 1. TNF- and UV treatment lead to serine phosphorylation of IB, whereas pervanadate and H/R induce tyrosine phosphorylation of IB. HeLa cells were treated with UV (50 J/m²), TNF- (10 ng/ml), pervanadate (50 µM) or H/R (5 hours hypoxia, 15 and 30 minutes reoxygenation) for the indicated times. Both untreated and treated samples were harvested for cytoplasmic extracts. (A) 5 µg of total protein was separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. Phosphorylation of IB at S32/36 was evaluated by a phosphospecific antibody. (B) 200 µg of total cytoplasmic protein was immunoprecipitated with anti-IB antibody followed by western blotting with an anti-phosphotyrosine antibody to detect tyrosine phosphorylation of IB. Samples analyzed were identical to those evaluated in A.