Recruitment of Sp1 to the cirp regulatory region at 32°C. HeLa cells (A) and BALB/3T3 cells (B-D) were cultured at 37°C, transferred to 32°C or remained at 37°C for 6 hours or indicated times, and then analyzed. (A) EMSA analysis of nuclear extracts using 32P-labeled MCRE oligonucleotide as a probe. comp, competition with a 50-fold molar excess of unlabeled wild-type (wt) or mutant (mt) probe. (+) and (−), present and absent, respectively. Arrow, specific probe-protein complex. Arrowhead, free probe. (B) Western blotting. Cell lysates were prepared at the indicated times, and analyzed using anti-Sp1 and anti-actin antibodies. (C) Immunofluorescence staining of cells. Sp1 was detected with anti-Sp1 antibody and FITC-conjugated anti-mouse IgG, and appears green under the confocal microscope. Nuclei were stained with DAPI, and appear blue. (D) ChIP assays. qPCR analysis for the 5′-upstream region containing MCRE and the first intron of the cirp gene using chromatins pulled down with anti-Sp1 antibody or anti-Sp3 antibody from cells incubated at the indicated temperatures. The amount of precipitated DNA relative to input (percent of input) was determined for each sample. Values are expressed as relative to those obtained in cells cultured at 37°C. Standard deviations from triplicate PCR reactions are indicated. Experiments were repeated three (A, B) or four (C, D) times, with similar results.