Chromatin immunoprecipitation (ChIP) assay
A3.01 cells were treated with RPMI culture medium containing 1% formaldehyde for 10 min. at 37degreesC. Cells were washed twice with ice-cold PBS and incubated for 10 min. on ice after resuspension in SDS lysis buffer (ChIP Assay Kit, Upstate). After centrifugation, pellets were resuspended in MNase reaction buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1 mM CaCl2, 4% NP-40). DNA digestion was performed using 50 U Micrococcal Nuclease (Fermentas) and 1 x 107 cells per tube in a volume of 1.5 ml. After 2 min, reaction was stopped by adding 30 microl 200 mM EGTA. Further steps were performed using the Chromatin Immunoprecipitation Assay Kit (Upstate) according to the manufacturer's instructions. 1 x 107 cells and 2 microg antibody (Sp1(Pep2) sc-59, Sp3(D-20) sc-644 or actin(H-196) sc-7210, Santa Cruz Biotechnology) were used for each immunoprecipitation. All buffers were freshly supplied with protease inhibitors. After phenol/chloroform extraction and ethanol precipitation, DNA was resolved in 16 microl H2O. Immunoprecipitated DNA was detected by nested PCR using 4 microl of the resolved DNA in the first PCR, and 1 microl for the nested PCR. For amplification of the A3G promoter, the following primers were used: ChIP3Gplus 5'-ccacggtggcctccgagggtga-3' and ChIP3Gminus: 5'-ctctccaccatcaagacagac-3' (1. PCR); ChIP3G2plus: 5'-tactctccctccctgtcccca-3' and ChIP3G nested minus: 5'-aggctgatgcctccgcag-3' (nested PCR). Taq polymerase (Qiagen) was used together with the following cycle conditions: one cycle 94degreesC for 2 min; 30 cycles 94degreesC for 30 s, 60degreesC for 60 s, 72degreesC for 2 min; one cycle 72degreesC for 10 min. As a negative control, a region in the A3G gene was targeted using the primers ChIP3Gneg_plus: 5'-taagtaccacccagagatgag-3' and ChIP3Gneg_minus: 5'-catgatcttcatggtggcacg-3' for both PCR steps. PCR conditions were the same as for the A3G promoter sequence, with the exception that annealing temperature was decreased to 55degreesC. 
