FIG. 3.
Effect of TNF-α on association between p47phox and TRAF4 in the cytoskeleton and membrane fractions of HMEC-1. (A) TRAF4 was immunoprecipitated (IP) from the Triton X-100-insoluble (cytoskeleton) fraction, and p47phox was then detected by immunoblotting (IB) in cells stimulated with TNF-α (30 min) either in the presence or absence of the PKC inhibitor Bis or chelerythrine (Chel). Immunoblotting for TRAF4 confirmed equal precipitation. (B) Assessment of translocation and binding to p22phox. p22phox was immunoprecipitated from the cellular membrane fraction, and p47phox was detected by immunoblotting. Protein extracted from U937 cells after stimulation with phorbol myristate acetate was used as a positive control. Similar results were obtained in two independent experiments.