Anti sumo2

Anti-HA monoclonal and polyclonal antibodies were purchased from Covance and GenScript, respectively. Anti-SUMO2 was from Life Technologies, anti-ubiquitin was from Santa Cruz Biotechnology, and anti-GAPDH antibody was from Millipore.

Cells were lysed in RIPA buffer (6 mM Na₂HPO₄, 4 mM NaH₂PO₄, 2 mM EDTA pH 8.0, 150 mM NaCl, 1% NaDOC, 0.1% SDS and 1% NP-40) and tissues were lysed in Tris–SDS buffer (2% SDS, 0.6 M Tris-Cl pH 8.0 and 0.1 M DTT) supplemented with protease inhibitors (Sigma-Aldrich) and 20 mM N-Ethylmaleimide (NEM; Sigma-Aldrich) and sonicated using the Diagenode Bioruptor. Lysates were clarified by centrifugation and protein concentration was measured using Protein Assay Dye reagent concentrate (Bio-Rad, Hercules, CA) according to the manufacturer’s protocol. The following antibodies and concentrations were used: anti-Flag M2; 1:7500 (Sigma-Aldrich), anti-LRH-1; 1:3000 for mouse liver and 1:10,000 for in vitro assay (R&D, Minneapolis, MN), anti-SF-1; 1:1000 (Upstate, EMD Millipore, Billerica, MA), anti-UBC9; 1:1000 (Cell Signaling, Danvers, MA), anti-SUMO1; 1:1000 (Developmental Studies Hybridoma Bank, Iowa City, IA), anti-SUMO2; 1:1000 (Life technologies) and Ubiquitin monoclonal P4D1 antibody; 1:1000 (Cell Signaling), HRP-conjugated anti-βactin 1:2500 (Cell Signaling), and anti-GAPDH 1:5000 (Santa Cruz Biotechnology, Santa Cruz, CA).