IL-2/α-CD40 reduces MMP2 and MMP9 expression and increases TIMP-1 and E-cadherin expression within the tumor microenvironment. (A) WT and GKO mice were treated as indicated. (B) WT mice were treated as indicated with or without continuous L-NAME treatment. On day 22, mice were euthanized and the primary tumor was dissected and homogenized in RIPA lysis buffer. The expression of MMP2, MMP9, TIMP-1, and E-cadherin was determined by Western blot analysis. HPRT was used as a loading control. The results are representative of at least three separate experiments. (C) For each protein, the corresponding band intensity was determined by densitometric analysis. The results were then divided by the band intensity for the corresponding HPRT loading control. For MMP9, the intensity of both major bands was analyzed together. The graphs represent the mean and standard errors for the quantitated results obtained from all experiments. *, P < 0.05; **, P < 0.005. The black bars indicate WT mice, the white bars indicate GKO mice, and the hatched bars indicate L-NAME–treated WT mice. (D) The expression of phosphorylated P53 (Ser 15) and total P53 levels was analyzed in treated samples by Western blot analysis. The graph illustrates the band intensity ratio of phosphorylated P53 relative to the corresponding total P53 levels from one experiment. The black and hatched bars indicate WT mice treated in the absence or presence of L-NAME, respectively.