FIGURE 2.

Interaction of SET7/9 with NF-κB p65 and requirement of SET7/9 methyltransferase activity for inflammatory gene regulation. A, SET7/9 does not affect IκBα degradation or p65 nuclear translocation upon TNF-α (10 ng/ml) treatment. Cytoplasmic and nuclear fractions prepared from SET7/9 knockdown THP-1 cells (SI) and control (CON) cells after treatment with TNF-α (10 ng/ml) for various time periods were subjected to immunoblotting (IB) with indicated antibodies. B, SET7/9 and p65 colocalize in the same cellular complex. HEK293 cells were co-transfected with SET7/9 and p65 expression vectors, and cell lysates were immunoprecipitated withβ-actin antibody (left) or p65 antibody (right). Immunopreciptates were immunoblotted with the indicated antibodies on the left. Representative of three separate experiments. C, HVSMC grown on coverslips were stimulated with TNF-α for 2 h, and p65 and SET7/9 were detected by immunofluorescent staining with p65 or SET7/9 antibody, followed by confocal microscopy. D, SET7/9 protein levels measured by Western blotting in control or SET7/9 knockdown THP-1 cells before and after TNF-α treatment. E, HEK293 cells were transiently transfected with an empty vector (CON) or vectors expressing wild type SET7/9 (wtSET7/9) or enzymatically inactive SET7/9 (mSET7/9). Basal and TNF-α (10 ng/ml, 2 h)-induced gene expression were determined by RT-PCR. F and G, quantification of data from E (^*, p < 0.001 methylation mutant of SET7/9 + TNF-α or SI + TNF-α versus CON + TNF-α, n = 3).