Figure 3.

Verification that CUP is an AP-2α isoform. (A) Immunoblot of recombinant hAP-2α1 and purified CUP. 50 μl of purified CUP (from fraction 29; Fig. 2A) or 10 ng of human rAP-2α1 were subjected to SDS/PAGE (10% acrylamide), after which the gel was transblotted onto a PVDF membrane and probed with rabbit anti-hAP-2α antibody. (B) EMSA of human rAP-2α1 (rAP2), day 7 adipocyte nuclear extract (A), or day 0 preadipocyte nuclear extract (P) with ³²P-labeled CUP-1 binding site oligonucleotide probe in the presence or absence of preimmune serum (PI), anti-hAP-2α antiserum (AP-2), anti-hSp1 antiserum (Sp-1), or unlabeled wild type (AP-2) or mutated (AP-2M) AP-2 binding site oligonucleotide. NE, nuclear extract. The single arrow identifies the CUP–oligonucleotide probe complex, and the double arrow identifies the position of the band supershifted by anti-hAP-2α antibody.