FIGURE 3.

TRAF6 required for SFK activation. HMVEC-Ls were transfected with TRAF6-targeting or control siRNA, and after 48 h they were processed for immunoblotting to detect TRAF6 protein (A) or after exposure to LPS (100 ng/ml) or medium alone a cell-based ELISA to detect SFK Tyr⁴¹⁶ phosphorylation (n = 4) (B). C, HMVEC-Ls transiently infected with increasing m.o.i. of Ad encoding for a FLAG-tagged TRAF6 DN were lysed and the lysates processed for FLAG immunoblotting. This blot is representative of two independent experiments. D, HMVEC-Ls infected with Ad-TRAF6 DN or Ad-null (m.o.i. = 100) were treated for 15 min with LPS (100 ng/ml) or medium alone, after which the cells were lysed and the lysates processed for phospho-SFK (Tyr⁴¹⁶) immunoblotting (n = 3). A, C, and D, to control for protein loading and transfer, blots were stripped and reprobed for β-tubulin. A, C, and D, IB, immunoblot; IB*, immunoblot after strip and reprobe. E, for each immunoblot generated in D, densitometric quantification of each phospho-SFK (Tyr⁴¹⁶) signal was normalized to β-tubulin signal in the same lane in the same blot. Vertical bars represent mean (±S.E.) arbitrary densitometry units of phospho-SFK signal normalized to arbitrary densitometry units of β-tubulin signal (n = 3). F, HMVEC-Ls were preincubated with cell-permeable TRAF6 decoy peptide or SP, after which they were exposed for 15 min to LPS (300 ng/ml) or medium alone, and the cells were processed for the cell-based ELISA to detect SFK Tyr(P)⁴¹⁶ (n = 9). * indicates significantly increased compared with the medium or siRNA control at p < 0.05. ** indicates significantly decreased compared with LPS + the control siRNA, Ad-null infected controls, or the control peptide at p < 0.05.