PMC:1920263 / 15340-17307 JSONTXT

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Further steps were performed using the Chromatin Immunoprecipitation Assay Kit (Upstate) according to the manufacturer's instructions. 1 × 107 cells and 2 µg 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 µl H2O. Immunoprecipitated DNA was detected by nested PCR using 4 µl of the resolved DNA in the first PCR, and 1 µl 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 94°C for 2 min; 30 cycles 94°C for 30 s, 60°C for 60 s, 72°C for 2 min; one cycle 72°C 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. 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