Fig. 2. Silencing p190B results in a decrease in MT1-MMP cell-surface presentation and MMP2 activation. (A) Cell lysates from HUVECs transfected with the indicated siRNAs and subsequently treated with PMA for 30 minutes were analysed by gelatin zymography. MMP2 was activated upon PMA treatment. The active form of MMP2 (62 kDa) became apparent, revealing the processing of pro-MMP2. In p190B-KD cells, MMP2 activation was decreased. No significant alteration of MMP2 activation was observed in the p190A-KD. The effect was quantified by measuring the percentage of active MMP2 relative to control conditions after PMA treatment. Each bar represents the mean±s.e. of three independent experiments. ^*P<0.005 in Student's t-test when compared with control. (B) Analysis of MT1-MMP expression in protein extracts from HUVECs transfected with the indicated siRNA by immunoblotting. β-actin expression was used as loading reference. Target knockdown was controlled using p190A- and p190B-specific antibodies. Specificity of the anti-MT1-MMP antibody was controlled using a previously described siRNA targeting MT1-MMP. (C) Quantification of MT1-MMP protein levels normalised using β-actin and relative to control (control-siRNA-transfected cells) is represented in the bar graph. Each bar represents the mean±s.e. (n=4 for A1 and B1, n=3 for A2 and B3). ^*P<0.001 in Student's t-test when compared with control. (D) MT1-MMP surface expression is altered in p190-KD cells. Biotinylated cell-surface proteins and non-biotinylated intracellular proteins were isolated from p190A- and p190B-KD cells and analysed by immunoblotting using anti-MT1-MMP, anti-β-actin and anti-β1-integrin antibodies. (E) The graph represents the quantification of four independent experiments. The amount of MT1-MMP present in cell-surface fractions significantly increased in p190A-KD cells and decreased in p190B-KD cells. ^*P<0.05 Student's t-test when compared with control.