RNA interference and western blot analysis
Sp1 and Sp3 translation was silenced in HeLa cells using the siRNA duplexes Hs_SP1_1_HP and Hs_SP3_1_HP (Qiagen). A nonspecific siRNA (Qiagen) was used as control. HeLa cells were transfected with 150 or 300 ng siRNA per 6-well, using the HiPerfect transfection reagent (Qiagen) according to the manufacturer's protocol for reverse transfection of adherent cells in 6-well plates.
Forty-eight hours after transfection, HeLa cells were harvested for detection of Sp1 and Sp3 proteins. Cells were washed in PBS, lysed in RIPA (25 mM Tris pH 8.0, 137 mM NaCl, 1% Glycerol, 0.5% sodium deoxycholate, 1% NP-40, 2 mM EDTA pH 8, 0.1% SDS and protease inhibitors) and lysates were cleared by centrifugation. After boiling with Laemmli′s buffer, samples were subjected to SDS–polyacrylamide gel electrophoresis followed by transfer to a nitrocellulose membrane. Sp1 and Sp3 proteins were detected using α-Sp1(Pep2) antibody (sc-59, Santa Cruz) or α-Sp3(D-20) antibody (sc-644, Santa Cruz) followed by incubation with α-rabbit-HRP (Amersham Biosciences). For detection of tubulin, α-tubulin (B5-1-2, Sigma) and α-mouse-HRP (Amersham Biosciences) antibodies were used. Signals were visualized by enhanced chemiluminescence (ECL, Amersham Biosciences).