Protein extracts from dox-treated A5 cells were immunoprecipitated with the indicated antibodies, followed by Western blotting. R, G, and M indicate rabbit, goat, and mouse, respectively, which were the source for the indicated antibodies. Asterisks indicate detection of IgGs by secondary antibody. (A) Co-IP showing the absence of P14ARF-SP1 interaction. (B) Co-IP verifying that P14ARF interacts with HDM2 in A5 glioma cells. P14ARF was immunoprecipitated with an anti-P14ARF antibody (C18). Input levels (right panels) for P14ARF were detected with an anti-HA tag (Y11R, R) antibody. (C) Co-IP assays demonstrating that P14ARF reduces HDM2-SP1 interaction. In the two left panels, immunoprecipitations with anti-HDM2 antibodies show that HDM2 interacts with SP1 and this interaction decreases when P14ARF is induced. This regulation is confirmed by reverse immunoprecipitation of HDM2-SP1 complexes with anti-SP1 antibody in the third panel. Western blotting of HDM2 and SP1 in the cell extracts (input) shows their expression is not affected by P14ARF induction (far right panel). Species-matched IgGs were used as controls. (D) Co-IP experiments showing that p19Arf silencing increases the endogenous Sp1-Mdm2 interaction. After siRNA silencing of p19Arf for 48 hours in mouse 3T3 fibroblasts, nuclear (Nuc) and cytosolic (Cyt) extracts were used for co-IP. First and second panels: Co-IPs using anti-Mdm2 and anti-Sp1 antibodies demonstrate that Mdm2 binding to Sp1 is increased in the nuclear fraction upon p19Arf silencing. Third panel: The co-IP with anti-p19Arf antibody confirms p19Arf interaction with Mdm2 in the nucleus. Fourth panel: Input levels of Sp1, Mdm2, and p19Arf in nuclear and cytoplasmic extracts.