Immunostaining and Immunoblot analysis
For immunostaining, cells were seeded at a density of 3–5 × 104 cells per well in 4-well chamber-slides and grown overnight at 33°C, unless indicated otherwise. Fixation was with ice-cold 4% formaldehyde in PBS, pH 7.4, for 20 minutes, or with ice-cold methanol/acetone (1:1) for 10 minutes at -20°C followed by air-drying (antibody-dependent). Cells were washed with PBS at least 2 times, 5 minutes per wash, between each of the following steps of the staining procedure: 0.1 M glycine in PBS for 5 minutes, 0.05% or 0.1% (antibody-dependent) Triton X-100 (Fisher Scientific) in PBS for 5 minutes, 2% bovine serum albumin (BSA) in PBS for 30 minutes, primary antibody diluted in 2% BSA/PBS for 90 minutes, secondary antibody diluted in 2% BSA/PBS for 60 minutes. All incubations were carried out at room temperature. Following staining procedures, slides were coverslipped with Vectashield mounting medium (Vector Laboratories) and analyzed on a BioRad Radiance 2100 confocal microscope (Biorad), with identical exposure settings for wild-type and mutant like images. All comparisons of wild-type and mutant staining were performed in Adobe Photoshop with identical brightness and contrast adjustments.
Total proteins were isolated from cell pellets by extraction with ice-cold 20 mM Tris, pH 7.4, 1% Triton X-100 (membrane-research grade, Roche), plus protease inhibitors (Complete mini tablet, 0.7 μg/ml pepstatin A, 2 μg/ml aprotinin, 5 μg/ml leupeptin [Roche]). Following homogenization through a 25-gauge needle (~10 passes), extracts were centrifuged at 1000 × g for 10 minutes, at 4°C, to remove debris. Typically, 20–40 μg of protein (determined by Bio-rad Dc Protein Assay) was separated by SDS-PAGE, for subsequent immunoblotting, as described [12]. 16.5% tris-tricine SDS-PAGE gels were used for subunit c immunoblotting experiments.