Human embryonic kidney (HEK 293T), HeLa, and COS-7 cells were obtained from ATCC (Manassas, VA, USA). Bovine serum albumin was obtained from Fisher Scientific (Loughborough, UK). Dulbecco's modified Eagle's medium (DMEM) and PBS were obtained from Invitrogen (Grand Island, NY, USA). Fetal bovine serum (FBS), l-glutamine, and penicillin-streptomycin were obtained from GIBCO (Grand Island, NY, USA). The liposomal transfection reagent FuGENE HD was purchased from Promega (Madison, WI, USA). The following were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA): endoglycosidase H (Endo H) (A0810), protease inhibitors (S8830, SIGMAFAST protease inhibitor cocktail tablets), phosphatase inhibitor cocktail (P0044), phosphatase inhibitor cocktail 2 (P5726), β-mercaptoethanol (BME) (M7154), glycerol (G5516), thapsigargin (T9033), dimethyl sulfoxide (DMSO) (5879), curcumin (C7727), and dithiothreitol (D9779). Turbo Pfu DNA polymerase was obtained from Stratagene (La Jolla, CA, USA). A bicinchoninic acid protein (BCA) assay kit (No. 23227) and sepharose A/G beads (No. 20421) were obtained from Pierce (Rockford, IL, USA). Immunofluor mounting media were purchased from Dako (Carpinteria, CA, USA).
Antibodies, their dilutions, and sources included the following antibodies for immunofluorescence (IF) and Western blot (WB): mouse FLAG epitope tag (DYKDDDDK) (anti-FLAG) monoclonal antibody (1:1000, IF and WB; Sigma-Aldrich Corp.), rabbit anti-FLAG antibody (1–2 μg/mL immunoprecipitation [IP], F7425; Sigma-Aldrich Corp.), rabbit anti-calnexin polyclonal antibodies (1:50, IF; Santa Cruz, Dallas, TX, USA), mouse monoclonal anti–ubiquitin (Ub) antibody (1:1000, WB, U0508; Sigma-Aldrich Corp.), AlexaFluor 568 goat anti-mouse IgG (1:200; Molecular Probes, Grand Island, NY, USA), and AlexaFluor 488 goat anti-rabbit IgG (1:2000; Molecular Probes). Secondary antibodies for WB were anti-rabbit IgG (whole molecule)-peroxidase antibody produced in goat (A0545, 1:30,000; Sigma-Aldrich Corp.), anti-mouse IgG (whole molecule)-peroxidase antibody produced in rabbit (A9044, 1:50,000; Sigma-Aldrich Corp.), and Pierce ECL plus WBsubstrate (32132; Thermo Scientific, Rockford, IL, USA).