6068 Nucleic Acids Research, 2008, Vol. 36, No. 19 CGGCGGGCTCCCCGTAC-30 and hC/EBPBR/EcoRI: 50-GAATTCCTAGCAGTGGCCGGAGGA-30. The fra- gments were veriﬁed by sequencing and subcloned into the pcDNA3/HA vector using BamHI and EcoRI. The sumoylation mutants of HA/LAP1, HA/LAP1K174A, and HA/LAP2, HA/LAP2K151A, were generated with a QuikChang site-directed mutagenesis kit (Stratagene, CA, USA). The reporters containing the COX-2 promoter ( 207/+49) and mutants were previously described (14). Expression vectors encoding Flag-HDAC1 and Flag- HDAC4 were generated by Dr Wen-Ming Yang (IMB, National Chung-Hsing University, Taiwan). Transfection and reporter gene assay Human epidermoid carcinoma A431 cells were grown in DMEM supplemented with 10% FBS, 100 mg/ml strepto- mycin and 100 U/ml penicillin. In this series of experi- ments, cells were cultured in serum-free condition with or without treatment with 25 ng/ml EGF. COX-2 reporter and indicated expression vectors were cotransfected by lipofection using the Arrest-In reagent or Lipofectamine 2000 according to the manufacturer’s instruction. The total DNA amount for each transfection was matched with pcDNA3 or related backbone vectors. After 6-h transfection, cells were changed in fresh serum-free DMEM medium and then were treated with EGF if neces- sary for 15 h. Small interfering RNAs assay Small interfering RNA (siRNA) pools for CEBPB and scrambled control siRNA were purchased from Ambion (Austin, TX, USA). The siRNA of HDAC4 was pre- viously described (37,38) and purchased from Invitrogen were (sense strands): HDAC4-1 (HD4-1), 50-AAUGUAC GACGCCAAAGAUTT-30 and HDAC4-2 (HD4-2), 50-G ACGGGCCAGUGGUCACUG-30. Cells were separately transfected with CEBPB siRNA and control siRNA by using Lipofectamine 2000. After 24 h of transfection, the lysates of transfectants were harvested after stimulation with or without EGF for 5 h for western blot with indi- cated antibodies. 10% glycerol and 1 mM dithiothreitol (DTT), under rota- tion at 48C for 3 h, and then plus protein A-agarose beads were added for an additional 2 h. Immunoprecipitated beads were washed three times with washing buﬀer, 1 PBS containing 0.1% IGEPAL CA-630. The immuno- complexes were separated by 10% SDS–PAGE, followed by western blot with anti-HA, anti-SUMO1 anti-SUMO2/ 3 or anti-HDAC4 antibodies. DNA affinity precipitation assay After 6 h serum-free starvation, nuclear extracts of EGF- treated A431 were harvested as indicated time courses. DAPA was performed according to a previously described method (14). Brieﬂy, the 200 mg of lysates extracted from each group were incubated with 2 mg of biotinylated C/EBP or CRE oligonucleotides in the presence of DNA binding buﬀer. After 1 h of incubation at 48C, 40 ml of streptavidin–sepharose were added to the reaction mixture and the incubation was continued for 1 h. The complexes were then precipitated by centrifugation and washed three times with DNA binding buﬀer before they were resolved by SDS–PAGE and subsequently analyzed by immuno- blotting with speciﬁc antibodies. The sequences of 50-bio- tinylated oligonucleotides were followed: CRE: 50-CACC GGGCTTACGCAATTTTT-30 and CEBP: 50 -CAGTCA TTTCGTCACATGGGC-30. Electrophoretic mobility shift assay An EMSA was performed essentially as described by Wang et al. (14). Brieﬂy, the 32P-labeled probes (0.2–0.5 ng) con- taining the individual CEBP or CRE site were incubated with 1 ml of in vitro-translated HA/LAP1, HA/LAP2 or HA/LIP in speciﬁc binding buﬀer containing 10 mM Tris–HCl (pH 7.5), 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 1 mg of poly(dI-dC) and 10% glycerol. After 20 min of incubation at room temperature, the reaction mixtures were resolved in 5% native polyacrylamide gels (acrylamide/bisacrylamide ratio, 30:1) at 48C, and the spe- ciﬁc protein–DNA complexes were visualized by autora- diography. The sequences of various oligonucleotides are as described above. In vivo SUMO modification assay and Co-IP assay Chromatin IP and re-chromatin IP assay A431 cells were transfected with the expression vectors of HA/LAP1, HA/LAP1K174A, HA/LAP2 or HA/ LAP2K151A in the presence or absence of SUMO1 expression vectors by using the Arest-In reagent. After 24-h transfection, cells were washed twice with phosphate buﬀered saline (PBS) containing 20 mM N-ethylmaleimide (NEM) and then harvested by scraping into 200 ml of RIPA buﬀer containing protease inhibitors as previously described (14). For the Co-IP assay, A431 were trans- fected with 2 mg of the indicated expression vectors by using Arest-In reagent. After 24 h, cells were harvested with modiﬁed RIPA buﬀer plus protease and phosphatase inhibitors which the details were previously described (14). Five hundred micrograms of cell lysate was immunopre- cipitated with 2 mg of anti-HA antibodies in IP buﬀer in a 1:4 ratio of modiﬁed RIPA and IP buﬀer, 20 mM HEPES (pH 7.9), 2 mM MgCl2, 0.2 mM EDTA, 0.1 mM KCl, The chromatin IP (ChIP) assay was performed essentially as described by Wang et al. (14). Brieﬂy, A431 cells were treated with 1% formaldehyde for 15 min. The cross- linked chromatin was then prepared and sonicated to an average size of 500 bp. The DNA fragments were immu- noprecipitated with antibodies speciﬁc to CEBPB, CEBPD, SUMO1, HDAC1, HDAC4 or control rabbit immunoglobulin G (IgG) at 48C overnight. After reversal of the cross-linking, the immunoprecipitated chromatin was ampliﬁed by primers related to speciﬁc regions of the COX-2 genomic locus. For the re-ChIP assay, the ﬁrst immune complex that had been washed twice with washing buﬀer of 50 mM Tris–HCl (pH 8.0), 0.1% SDS, 0.5% NP-40, 150 mM NaCl and 2.5 mM EDTA, and three times with low-salt buﬀer of 10 mM Tris–HCl (pH 8.0) and 0.1 mM EDTA, was then resolved in 10 mM DTT at 378C, further diluted in ChIP dilution buﬀer and then