6474 Nucleic Acids Research, 2006, Vol. 34, No. 22 cells were prepared as described previously (27,28) and kept frozen at 80 C until use. The NFI-L-enriched rat liver carboxymethyl (CM)-Sepharose fraction has been described previously (29). The protein concentration was determined for each nuclear extract by the Bradford procedure and further validated by Coomassie blue staining of SDS–poly- acrylamide fractionated nuclear proteins. Double-stranded synthetic oligonucleotides corresponding to: (i) the p21 pro- moter region located from 167 to 127 and bearing the target region for NFI (p21.1: 50-GCCTGCTGGAACTCGG- CCAGGCTCAGCTGGCTCCGCGCGG-30), (ii) the high- afﬁnity binding site for CTF/NFI (50-GATCTTATTTTGG- ATTGAAGCCAAT-ATGAG-30) or (iii) the 80 bp HindIII– SmaI fragment from p21 to 192 were 50 32P-end-labeled as described (30) and used as probes in the electrophoretic mobility shift assays (EMSAs). Approximately 3 · 104 c.p.m. labeled DNA was incubated for 5 min at room temp- erature with nuclear extracts in the presence of poly(dI:dC) (Amersham) and 50 mM KCl in buffer D (10 mM HEPES, 10% v/v glycerol, 0.1 mM EDTA, 0.25 mM phenylmethylsul- fonyl ﬂuoride). DNA–protein complexes were separated by electrophoresis on native 4 or 6% polyacrylamide gels run in Tris/glycine buffer at 4 C, as described previously (30). Competition experiments in EMSA were conducted as described above except that up to 500-fold molar excesses of the following unlabeled double-stranded oligonucleotides were added to the reaction mixture as unlabeled competitors: p21.1, or derivatives of p21.1 that bear mutations (bold, small letters) into either the 50 (in p21.1m50: 50-GCCTGCT- aaAACTCGGCCAGGCTCAGCTGGCTCCGCGCTGG-30) or the 30 NFI half-site (in p21.1m30: 50-GCCTGCTG- GAACTCGattAGGCTCAGCTGGCTCCGCGCTGG-30), or mutated into both half-sites (in p21.1m5030: 50-GCCTGC- TaaAACT-CGattAGGCTCAGCTGGCTCCGCGCTGG-30), p21.2 (50-GCCTGCTGGAACTCGGCCAGGC-TC-30) and p21.3 (50-CAGCTGGCTCCGCGCTGG-30) which corre- spond to each half of the p21.1 sequence, CTF/NFI, AP1 (50-GATCCCCG-CGTTGAGTCATTCGCCTC-30) and Sp1 (50-GATCATATCTGCGGGGCGGG-GCAGACACAG-30). EMSA supershift experiments were conducted as above except that 3 ml of either a pre-immune serum, or a polyclonal antibody that can recognize all isoforms of NFI (sc-5567, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was incu- bated with the proteins for 5 min prior to the addition of the probe. Methylation interference The 83 bp SmaI–HindIII fragment from the p21–192 plasmid that covers p21 promoter sequences from 110 to 192 was 50 end-labeled at the 110 position (top strand) and partially methylated with DMS as described (31). Approximately 3 · 105 c.p.m. methylated, labeled probe was incubated with 100 mg crude nuclear proteins from HeLa cells in the pres- ence of 1 mg poly(dI:dC) in buffer D and DNA–protein com- plexes were separated by electrophoresis through a 6% native polyacrylamide gel in Tris-glycine buffer. Following electro- phoresis, the DNA–protein complexes were visualized by autoradiography and isolated by electroelution as described (32). The isolated labeled DNA was then treated with piper- idine (33) and further analyzed on a 6% sequencing gel. DNase I in vitro footprinting The 228 bp XbaI/KpnI DNA fragment spanning the p21 pro- moter sequence from position 192 to +36 was 50 end- labeled and used as a probe in DNase I footprinting. DNase I digestion was performed in DNase I buffer (34) by incubat- ing 4 · 104 c.p.m. labeled probe with 75 mg crude nuclear proteins from HSF cells. Further analysis of the digested products onto polyacrylamide sequencing gels was done as described previously (35). SDS–PAGE and Western blot One volume of sample buffer (6 M urea, 63 mM Tris [pH 6.8], 10% [vol/vol] glycerol, 1% SDS, 0.00125% [wt/ vol] bromophenol blue and 300 mM b-mercaptoethanol) was added to 15 mg proteins before they were size fraction- ated on a 10 or 12% SDS–polyacrylamide minigel and electro-transferred onto a nitrocellulose membrane o/n at 4 C. The blot was then washed once (5 min) with TS buffer (150 mM NaCl and 10 mM Tris–HCl [pH 7.4]) and once (30 min) in TSM buffer (TS buffer plus 5% [wt/vol] fat- free powdered milk and 0.1% Tween-20). Then, a 1:5000 dilution of the NFI antibody in TSM buffer was added to the membrane and incubation proceeded for at least 90 min at 22 C. The blot was then washed four times in TSM buffer and incubated an additional 90 min at 22 C in a 1:5000 dilution of a peroxidase-conjugated goat anti-rabbit immunoglobulin G (Jackson Immuno-research, Biocan Scientiﬁc, Mississauga, ON, Canada). The membrane was successively washed in TSM (twice, 5 min each) and TS (twice, 5 min each) before immunoreactive complexes were revealed using western blot chemiluminescence reagents (Renaissance, NEN Dupont, Boston, MA) and autoradiographed. Transient transfections and CAT assay Primary cultured HSFs and the established tissue culture cell lines HeLa and GH4C1 were all transiently transfected with the p21-CAT recombinant constructs (p21–2300, p21–192 and its mutated derivatives p21–192 mNFI, p21–124) by the calcium phosphate precipitation method as described (36), using 15 mg of the test plasmid and 5 mg of the human growth hormone (hGH) gene-encoding plasmid pXGH5 (37). CAT activities were measured as described pre- viously (38) and normalized to hGH secreted into the culture medium, which was assayed using a radioimmunoassay kit (Medicorp, Montre´al, Que´bec, Canada). Each single value is expressed as 100 · (% CAT in 4h)/100 mg protein/ng hGH. To be considered signiﬁcant, each individual value needed to be at least 3-fold the value of the background level caused by the reaction buffer used (usually correspond- ing to 0.15% chloramphenicol conversion). To compare the groups, Student’s t-test was performed. Differences were considered to be statistically signiﬁcant at P < 0.05. All data are expressed as mean ± SD. Chromatin immunoprecipitation assays The ChIP analyses were conducted as previously described (39). Brieﬂy, HSFs were grown on 150-mm tissue culture dishes to 75% conﬂuence in complete DMEM, or in FBS- depleted DMEM for periods of time ranging from 48 to 72 h.