Cell Culture, Immunoprecipitations and Biochemistry

HEKtsa cells ([13] via T. Hoshi lab, University of Pennsylvania) and Cos-7 cells (ATCC #CRL-1651 via J. Trejo lab, UCSD) were maintained in growth media containing Dulbecco’s modified Eagles media (DMEM, Mediatech) supplemented with 10% fetal bovine serum (Omega), 1% penicillin/streptomycin (Mediatech) and 1% L-glutamine (Sigma) at 37°C with 5% CO₂. All experiments were performed using HEKtsa cells, except Shaker and Dα3 co-immunoprecipitations, which were performed using Cos-7 cells. Cells were transfected using X-tremeGene HP (for HEKtsa) or X-tremeGene-9 (for Cos-7) transfection reagent (Roche) as previously described [14]. Cells were lysed in SDS lysis buffer [10 mM Tris, pH 7.5; 100 mM NaCl; 5 mM EDTA; 1% Triton X-100; and 0.05% SDS with cOmplete protease inhibitor (Roche)] and prepared for immunoprecipitation or Western blotting as previously described [14]. α4-HA surface labeling was performed as previously described [12] using rabbit anti-HA (0.6 μl/ml Rockland) antibodies. For co-immunoprecipitation experiments, rabbit anti-GFP antibody (0.2 μl/ml Life Technologies) was used for Shaker and rabbit anti-HA (Rockland) was used for Dα3. Rabbit anti-GFP (1:500, Life Technologies), mouse anti-HA (1:1000, Covance) and mouse anti-MYC (1:250, Santa Cruz Biotechnologies) were used for Western blotting. Peptide-N-Glycosidase F (PNGase F) treatment of cell lysates to remove glycosylation modifications were performed according to the manufacturer’s instructions (PNGase, NEB) with the following modification: cOmplete Protease Inhibitor (Roche) was included during the enzymatic treatment (0.3 μl PNGase) which was performed for 2 hours at 37°C. PNGase-treated samples were run on a 12% gel (NuPage, Life Technologies) to maximize band separation at low molecular weight.