Mouse anti gfp 7 1 13

Cells were collected by centrifugation, washed once with water and disrupted by bead beating with a FastPrep 24 (MP Biomedicals) in 50 mM Tris-HCl pH 7.5 containing 0.5 mM EDTA, 1 mM PMSF and complete protease inhibitors (Roche). Proteins were solubilized by addition of 1.5% SDS and incubation at 65 C for 5 min. Lysates were cleared at 16,000 g at 4 C for 2 min and protein concentrations were determined with the BCA assay kit (Thermo Scientific Pierce). Equal amounts of protein were resolved by SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were probed with primary and HRP-coupled secondary antibodies and incubated with homemade ECL. Chemiluminescence was detected with an ImageQuant LAS 4000 imaging system (GE Healthcare). Images were quantified with ImageJ and processed with Adobe Photoshop. Antibodies were mouse-anti GFP 7.1/13.1 (Roche), mouse anti-mCherry 1C51 (Abcam) for detection of Luciferase-mCherry, rabbit anti-mCherry (Biovision) for detection of R-mCherry-sfGFP, rabbit anti-Sec61 (Peter Walter, UCSF), mouse anti-Pgk1 22C5 (Abcam), mouse anti-Pho8 1D3A10 (Abcam), mouse anti-FLAG M2 (Sigma), mouse anti-HA 6E2 (Cell Signaling) for detection of CFTR-HA, and rat anti-HA 3F10 (Roche) for detection of HA-tagged Roq1.