TIMs mediate MMP-9 induction by anti–VEGFR-2 therapy. Tumors were harvested at day 14, and mRNA expression was quantified using RT-PCR for Mmp9 (A) and Vegf-a (B). Relative mRNA expression was normalized to β-actin (n ≥ 5/group). (C) MMP-9 protein levels from tumors were analyzed by Western blot and normalized to α-tubulin (n = 6/group). Representative images are shown. (D) The same tumor lysates were analyzed by gelatin zymography (n = 6/group). Representative images are shown. Tumor tissue was subjected to histologic analysis of MMP-9 expression with DAPI (E). Scale bars represent 50 μm.