FIGURE 8.

p16 interacts with Sp1 through the fourth ankyrin repeat. A, p16-negative U2OS cells were transfected with the indicated constructs, and then whole cell extracts were prepared and used for immunoprecipitation (IP) with anti-p16 antibody (mouse IgG was used as control), and immunoblotting was performed utilizing the indicated antibodies. B, whole cell extracts were prepared from U2OS expressing the indicated constructs, and immunoblotting was performed for the indicated antibodies. C, ChIP assay. Chromatin was purified from the indicated cells and then immunoprecipitated using anti-Sp1 antibody. miR-141 and miR-146b-5p promoters were amplified by qPCR using specific primers. D, total RNA was prepared from U2OS expressing the indicated constructs and used to assess the level of the indicated genes by qRT-PCR using GAPDH as internal control. Error bars represent means ± S.D. E, U2OS cells were transfected with constructs bearing the indicated tumor-associated point mutations in the fourth ankyrin repeat of human p16. Whole cell extracts were prepared and used for immunoprecipitation with anti-p16 antibody (mouse IgG was used as control), and immunoblotting was performed utilizing the indicated antibodies.