Tests of EKLF acetylation by CBP, p300, and P/CAF in vitro. (A) COS7 cells were transfected and extracts were immunoprecipitated with the indicated antibodies. IP-HAT assays utilized 5 μg of GST-EKLF(76–376). Samples were electrophoresed and the dried gel was exposed in autoradiography (Upper). GST-EKLF(76–376) alone was tested for autoacetylation in lane 9. Locations of p300/CBP, P/CAF, and GST-EKLF are indicated. Protein samples were also stained to show that equivalent amounts were used (Lower). (B) Endogenous CBP from COS7 cells was immunoprecipitated, and IP-HAT assays were performed with 5 μg of various GST-EKLF fusion proteins as diagrammed on the Right, which also shows the locations and sequences of lysines conserved between mouse and human EKLF [amino acid residues 47, 74, 177, 279, 288; mouse numbering is based on initiator methionine being residue 19 (9)]. Proteins were resolved and subjected to autoradiography (Upper Left) or stained for protein (Lower Left). Asterisks show the location of nondegraded GST-EKLF fusion proteins. Molecular mass markers (on the left) are 70, 55, 33, 25, and 15 kDa (top to bottom).