Virus production and infection
Adenoviral particles were produced by Welgen, Inc. (Worcester, MA, USA) from a vector encoding TAP–LL5β. C2C12 myotubes were infected with adenovirus on the second day after fusion induction and collected 2 days later. Lentiviral and retroviral particles were obtained from HEK-293 and GP2 cells respectively. Packaging cells were transiently transfected with plasmids. Media were replaced 24 h later, then collected 24 or 48 h later, centrifuged at 6000 rpm for 10 min and passed through a 0.45 µm filter. For infection, virus-containing medium was mixed with fresh medium containing Polybrene (Santa Cruz, CA, USA; SC-134220; 8 µg/ml).
Complex purification and mass spectrometry analysis
For complex purification, myotubes infected with TAP-GFP-LL5β adenovirus, or control (uninfected) myotubes, were washed with ice cold PBS containing sodium azide and covered with lysis buffer [50 mM Tris-HCl, 150 mM NaCl, 50 mM NaH2PO4, 10 mM imidazole, 0.1% Nonidet-P40, 10% glycerol, 10 mM β-mercaptoethanol, EDTA-free Mini protease inhibitor cocktail (Roche, Indianapolis, IN, USA), 1 mM phenylmethylsulphonyl fluoride; pH 8.0]. Cells were scraped off the dish, incubated briefly on ice, passed 3 times through a 25 gauge needle with syringe and centrifuged for 5 min at 4000 rpm and 30 min at 21000 rpm. The supernatant was incubated with washed IgG Sepharose 6 Fast Flow from GE Healthcare (Waukesha, WI, USA; 17-0969-01) for 4–16 h. Next, the Sepharose was loaded into a column (Bio-Rad, Hercules, CA, USA; 731-1550), washed 3 times with wash puffer (50 mM Tris-HCl, 50 mM NaH2PO4, 150 mM NaCl, 0.1% NP-40; pH 8.0), washed once with TEV buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5 mM EDTA, 1 mM DTT; pH 8.0) and resuspended in TEV buffer. To cleave the protein from the Sepharose, AcTEV (Invitrogen, Grand Island, NY, USA; 12575-015) was added and the sample was incubated for 2 h at room temperature or overnight at 4°C. For precipitation of proteins from eluted samples, 25% of the sample volume of 100% TCA (500 g TCA in 350 ml of H2O) was added, incubated for 10 min at 4°C and centrifuged at 14000 rpm for 5 min. The pellet was washed with 200 µl of cold acetone, centrifuged at 14000 rpm for 5 min, air dried and resuspended in sample buffer followed by incubation at 95°C for 6 min. For analysis, samples were subjected to SDS-PAGE electrophoresis and proteins were visualised with SilverQuest (Invitrogen, Grand Island, NY, USA; LC6070). Gels were stained with Colloidal Blue Staining Kit (Invitrogen, Grand Island, NY, USA; LC6025). Slices were excised from the gel and analysed at the Harvard Microchemistry and Proteomics Analysis Facility by microcapillary reverse-phase HPLC nano-electrospray tandem mass spectrometry (μLC/MS/MS) on a Thermo LTQ-Orbitrap mass spectrometer.
C2C12 cells were obtained from American Type Culture Collection (Manassas, VA, USA; CRL-1772). Cells were cultured for 5 or fewer passages in DME containing 20% foetal calf serum supplemented with glutamine, penicillin, streptomycin and Fungizone. Cells were trypsinised and replated onto 15 cm cell culture dishes. Before plating, dishes were coated with 10 µg/ml solution of laminin 111 (Invitrogen, Grand Island, NY, USA; 23017-015) in L-15 medium supplemented with 0.2% NaHCO3, incubated overnight at 37°C, and aspirated immediately before plating cells. To induce cell fusion, growth media was replaced with fusion media containing 2% horse serum in DMEM supplemented with glutamine, penicillin, streptomycin and Fungizone.
HEK293 and HeLa cells were from American Type Culture Collection (Manassas, VA, USA; CRL-1573 and CCL-2, respectively) and were cultured in DMEM (Dulbecco's modified Eagle's medium, Lonza) containing 10 % FBS (Eurx), 1% glutamine and 1% penicillin/streptomycin (Life Technologies). Transfection of HEK293 and HeLa cells was performed using Lipofectamine 2000 (Invitrogen; 11668027) according to the manufacturer's instructions.
HEK-293 cells were transfected with appropriate constructs, washed with ice-cold PBS and covered with lysis buffer [50 mM Tris-HCl, 150 mM NaCl, 0.1% Nonidet-P40, 10% glycerol, 1 mM DTT, EDTA-free Mini protease inhibitor cocktail (Roche, Indianapolis, IN, USA); pH 8.0]. Cells were scraped off the dish, incubated briefly on ice, pipetted vigorously, and centrifuged for 30 min at 21000 rpm. Supernatants from centrifugations were incubated overnight with Dynabeads (Invitrogen, Grand Island, NY, USA; 658-01D) coated with anti-GFP antibody (Invitrogen, A-11120). Beads with attached proteins were washed four times with lysis buffer, resuspended in 2×SDS sample buffer and boiled for 5 min. For western blot analysis, samples were loaded on 10% acrylamide gels, subjected to the SDS-PAGE electrophoresis and transferred to the Biotrace NT nitrocellulose membrane (Pall; Port Washington, NY, USA). Anti-FLAG antibody (clone M2; Sigma, St. Louis, MO, USA; F1804-200UG) was used to detect FLAG-LL5BIP and the anti-GFP antibody used for western blot was from Epitomics (Burlingame, CA, USA; 1533-1).
Mouse LL5BIP ORF (sequence ID BC016099) was amplified by PCR from C2C12 cDNA, previously generated from RNA isolated from C2C12 myotubes using High Capacity cDNA Reverse Transcription Kit (Life Technologies; Carlsbad, CA, USA). The primers used were: ATGGCTGAGAG (forward) and CTAGTCATGACGCAC (reverse) with appropriate overhangs for restriction enzyme digest. The PCR products were purified and cloned into FseI and AscI sites of pCDNA3.1/FFT-N/Puro, a custom-made plasmid based on the pCDNA3.1 backbone with the sequence of two tandem FLAG tags and a TEV restriction site upstream from the multiple cloning site (MCS) and FseI and AscI restriction sites engineered into the MCS. The resulting construct was FLAG-LL5BIP. Cloning of GFP-LL5β was described previously.