Figure 6:

SIAH2 forms a complex with ZFP521 and EBF1. (A – C) HEK293T cells were transfected with equal amount of cDNA encoding wild-type Flag-SIAH2 (Flag-SIAH2^wt), dominant negative mutant Flag-SIAH2 (Flag-SIAH2^dnm), HA-ZFP521 or EBF1. pcDNA 3.1 was used to balance out the total cDNA amount transfected. Flag-SIAH2^dnm represents a H⁹⁹A-C¹⁰²A (RING mutant) form of SIAH2 that abolished SIAH2 ligase activity. After 48 hours transfection, cells were treated with 10 μM MG132 for 3.5 hours and then treated with 25 μM PR619 for an additional 30 minutes. (A) Cells were collected and analyzed by western blotting, representing 10% of input samples used in (B) and (C). Cell lysates were affinity purified using (B) anti-FLAG M2 antibody or (C) anti-hemagglutinin antibody. pCMV-GFP was used as an internal control for transfection efficiency. (D-E) 3T3-L1 preadipocytes were induced for adipocyte differentiation at the indicated time points. (D) Cells were collected and analyzed by western blotting, representing 10% of input samples used in (E). (E) Cell lysates were affinity purified using a rabbit anti-SIAH2 or a nonspecific rabbit anti-IgG antibody as an isotype control. β-actin is included as a loading control. IB, immunoblot; IP, immunopurified. All experiments were performed at least twice.