Fig. 4. TAFI68 is acetylated in vitro and in vivo. (A) PCAF acetylates cellular SL1 in vitro. Immunopurified SL1 (lane 1), recombinant p53 (lane 2) and BSA (lane 3) were incubated with 500 ng of FLAG-PCAF and 1 µCi of [3H]acetyl-CoA, separated by 10% SDS–PAGE and acetylated proteins detected by fluorography. Autoacetylated PCAF is indicated. (B) TAFI68 is acetylated by the cellular PCAF complex. FLAG-tagged PCAF isolated from transfected NIH-3T3 cells (lane 1) or 500 ng of recombinant FLAG-PCAF purified from Sf9 cells (lane 3) was used to acetylate recombinant TAFI68 that was expressed in Sf9 cells (lane 2). Acetylation was monitored on immunoblots with α-acetyl-lysine antibodies. A Coomassie Blue stain of TAFI68 is shown on the left. (C) TAFI68 is acetylated in vivo. TIF-IB/SL1 was immunopurified from PC-1000 fractions (Schnapp and Grummt, 1996) from Ehrlich ascites (lane 1) and HeLa cells (lane 2), and subjected to western blotting using α-acetyl-lysine antibodies (lanes 3 and 4). In lanes 1 and 2, the western blot was reprobed with α-TAFI95 and α-TAFI68 antibodies.