Anti ha 11 clone 16b12

One-step immunoprecipitations of membrane proteins were performed using aminolink plus protein A/G resin (Pierce) with covalently bound anti-CD81 (clone 1.3.3.22, Santa Cruz), anti-HA.11 (clone 16B12, Covance) or IgG₁κ isotype control (clone MOPC-21, BD Pharmigen) antibodies. CD81 and associated proteins were eluted from the resin using glycine buffer (pH 2.8) and precipitated with ethanol, sodium acetate (pH 5) and glycogen as described elsewhere [86 (link)]. Experiments were conducted in four biological replicates from four independent hepatoma cell passages. Primary human hepatocyte co-IPs were performed as single experiments from seven independent donors. Efficiency of bait enrichment was determined for each hepatoma cell IP sample and for IPs from two primary cell donors by immunoblotting. All hepatoma cell IP eluates and IPs from primary cells of five independent donors were used for liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis.

MCF7 cells were originally provided by Marvin Rich (Karmanos Cancer Institute, Detroit, MI, USA), and cultured in improved minimal essential media (IMEM) supplemented with 5% fetal bovine serum (FBS). The pSG5 plasmid vector with cDNA insert encoding wild type, hemagglutinin (HA)-tagged murine ERRγ (100% identical to human ERRγ) has been described previously [19 (link),20 (link),33 (link)]. MCF7 cells were transiently transfected with HA-ERRγ or pSG5 empty vector for 27 h using JetPrime (VWR, Radnor, PA, USA) prior to whole cell lysis, polyacrylamide gel electrophoresis, protein transfer to nitrocellulose membranes, immunoblotting, and chemiluminescent detection performed as described in [20 (link),34 (link)]. Primary antibodies used were: anti-HA.11 clone 16B12 at 1:500 (Covance, Princeton, NJ, USA); anti-EEF1A2 SAB2100650 at 1:500 (Sigma, St. Louis, MO, USA); and anti-PPIF SAB4500035 at 1:500 (Sigma). Membranes were reprobed for β-actin (Sigma, 1:10,000) as a loading control. NIH ImageJ (http://rsbweb.nih.gov/ij/) was used for densitometric analysis of ERRγ (HA), EEF1A2, and PPIF expression relative to β-actin. Levels of ESRRG, EEF1A2, and PPIF mRNA in MCF7 and BT-474 cell line samples published in [35 (link),36 (link)] were obtained from ONCOMINE [37 (link)].