Figure 5
Effect of IL-32β on the activation of NF-κB and STAT3 in tumor tissues, and cultured colon and prostate cancer cells. (a) The DNA-binding activity of NF-κB was determined by EMSA in the nuclear extracts of tumor tissues of IL-32β-overexpressed transgenic mice or the athymic nude mice (a, upper panel). Expression of p50 and p65 in nuclear extracts (NE, middle panel of a), IκB phosphorylation in the cytosol extracts (CE, lower panel of a), and DNA-binding activity and STAT3 phosphorylation (c) in total lysates of tumors were determined by EMSA (upper panel) or by western blotting (lower panel). (b and d) Expression of p65 (b), and phosphorylated STAT3 (d) in murine tumors were determined by immunohistochemistry. (e) Cellular localization p-STAT3 (green) and p65 (red) in tumor tissues. IL-32β transgenic and athymic nude mice were observed by fluorescence microscopy after Immunofluorescence staining, as described in Materials and Methods section. Each image and band are representative of three independent mice. (f) Colon and prostate cancer cells were transfected with the vector or the IL-32β and cultured for 24 h, and then NF-κB was determined by EMSA (f, upper panel). Expression of p50 and p65 in nuclear extracts (NE, middle panel) and IκB phosphorylation in the cytosol extracts (CE, lower panel) were determined by western blotting. (g) Phosphorylation of STAT3 in total cell extracts was analyzed by western blotting (g, left panel). Cellular localization of p-STAT3 (green) and p65 (red) in IL-32β-transfected colon and prostate cancer cells was determined with confocal microscopy, as described in Materials and Methods (f, right panel). Each band is representative of three independent experiments
