Morpholino-antisense oligomers for gp91 phox block VCAM-1 activation of endothelial cell MMP-2 and MMP-9. mHEVc cells were transfected with carboxyfluorescein-labeled control morpholino oligomers and the carboxyfluorescein-labeled gp91 phox morpholino antisense oligomers. The cells were cultured for 3 days to allow for turnover of previously synthesized gp91 phox, stimulated with anti–VCAM-1–antibody-coated beads, washed at the times indicated, and scraped into lysis buffer; equal amounts of protein were examined by zymography. (A) Flow cytometry verifies the transfection of the mHEVc cells with the carboxyfluorescein-tagged morpholinos. (B) gp91 phox protein expression. Western blots were performed with equal protein loading (80 μg/lane) and using mouse antimouse gp-91phox (a kind gift from Drs D. Roos and E. van der Schoot, University of Amsterdam, The Netherlands)²⁸  (1:200) followed by an HRP-conjugated rabbit antimouse secondary antibody (Amersham) and ECL detection. gp91 phox in the mouse has an apparent molecular mass of 58 kDa.²⁹  Lanes from a representative Western blot are above the graph. The Western blots were analyzed with Image J software from NIH. Only 2 nonspecific bands (200-230 kDa) (data not shown) were present and exhibited similar intensity in all lanes as previously reported.³⁰  (C) MMP-9 activity. (D) MMP-2 activity. (B-D) Data presented are the mean ± SD from 3 experiments. *P < .05 compared with mHEVc cells at 0 minutes. **P < .05 compared with control morpholino-oligomer–transfected mHEVc cells.