PMC:549398 / 4530-5474 JSONTXT

{"project":"2_test","denotations":[{"id":"15701759-10371501-77060029","span":{"begin":637,"end":639},"obj":"10371501"}],"text":"Cells, transfections and chloramphenicol acetyltransferase (CAT) assays\nThe 293T and HeLa cell lines were maintained in DMEM supplemented with 10% fetal bovine serum. In general, between 1 and 3 μg of DNA was transfected into 293T and/or HeLa cells using lipofectamine 2000 (Invitrogen) according to the manufacturer's protocols. PcDNA3 plasmid was used to equalize the amount of DNA for each transfection. Forty-eight hours post-transfection, the cells were harvested, washed with phosphate-buffered saline and then re-suspended in 50–100 μl of 0.25 M Tris, pH 7.8. The cell extracts, CAT assays were performed as described previously (14). To create stable Sam68 knockdown cell lines, HeLa cells were transfected with pSam68-RNAi and placed under G418 selection. The Sam68 expression in G418-resistant colonies was analyzed by western blot analysis using anti-Sam68 antibodies. A similar strategy was used to create HeLa-IL10i control clones."}