Sp1/3 cellular versus nuclear levels and in vivo occupation of the PDPN promoter. A) Immunoblot analysis and densitometric quantification of Sp1 and Sp3 protein contents in MG63 and Saos-2 cell lysates and nuclear extracts. Total cellular protein and nuclear extracts from MG63 and Saos-2 cells were resolved on 10% SDS-PAGE and blotted onto nitrocellulose. Levels of endogenous Sp1 and Sp3 proteins were determined by probing the blots with anti-Sp1 and anti-Sp3 antibodies, respectively. Sp1 shows a 105 kDa band, whereas Sp3 shows two bands at 120 and 75 kDa. Blots were reprobed with anti-GAPDH and anti-TFIID antibodies as loading control. After densitometric evaluation, Sp1/Sp3 levels were normalized to respective GAPDH and TFIID levels from the same blot. Data are given as the mean ± S.D. of samples of the experiment repeated three times. B) and C) Association of Sp1 and Sp3 to the PDPN promoter in vivo. Formaldehyde crosslinked chromatin was precipitated with an antibody specific for Sp1 (panel B) and Sp3 (panel C). PCRs were performed with primer pairs flanking the four Sp binding sites. L, 100 bp DNA ladder; +, immunoprecipitation with anti-Sp1 or anti-Sp3 antibody; co, precipitation control with unspecific rabbit IgG; -, negative control without antibody; Input, dilution series of control input DNA; no DNA, PCR negative control.