FIGURE 11.

TSP1 increases EGFR Tyr-1068 phosphorylation through MMP9 activation. A, A431 cells were preincubated with anti-MMP9 neutralizing antibody or a species- and isotype-matched antibody control, B7-1, after which the cells were treated for 0.5 h with TSP1 (214 nm) or media alone. Cells were lysed and the lysates processed for phospho-EGFR (Tyr-1068) immunoblotting. To indicate protein loading and transfer, blots were stripped and reprobed with anti-β-tubulin antibody. B-D, A431 cells were transfected with control or MMP9-targeting siRNAs after which they were incubated with TSP1 (214 nm, 0.5 h), E123 (214 nm, 1 h), EGF (1.67 nm, 10 min), or media alone. B, supernatants were concentrated 10-fold through ultrafiltration, and the concentrate supernatants were assayed for MMP9 by ELISA. Vertical bars represent mean (±S.E.) MMP9 levels in ng/ml (n = 6). ^*, indicates significantly increased compared with control siRNA-transfected media control at p < 0.05; ^**, indicates significantly decreased compared with the control siRNA-transfected cells with stimulus at p < 0.05. C, cells were lysed and the lysates processed for phospho-EGFR (Tyr-1068) immunoblotting. To indicate protein loading and transfer blots were stripped and reprobed with anti-β-tubulin antibody. D, cells were lysed and the lysates processed for phospho-PLCγ (Tyr-783) immunoblotting. To indicate protein loading and transfer, blots were stripped and reprobed with anti-PLCγ antibodies. A, C, and D, IB, immunoblot; IB^*, immunoblot after stripping. These blots are representative of ≥2 independent experiments.