Western Blotting

Western blot analysis was performed as previously described (20–25, 27). We grew PHBAE cells that expressed WT and mutant F508del-CFTR and CFBE41o cell lines to confluence in a monolayer. Briefly, we prepared whole-cell extracts in a 1% NP-40 lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 1 μM aprotinin, 2 μM leupeptin, and 1 μM pepstatin, 1 μM phenylmethylsulfonyl fluoride, 1 mM DTT, and 2 μM Na₃VO₄). We then recovered insoluble material, which we sheared by passing it through a 25-gauge needle. We used a BCA protein assay kit (Thermo Scientific) to quantitate protein by the Lowry assay.

We fractionated 100 μg of protein on a 6% SDS polyacrylamide gel under reducing conditions. We then transferred the fractionated proteins to nitrocellulose membranes and blocked blots in Tris buffered saline (Tween 20), which contained 5% nonfat dried milk. We probed the blots with a dilution (1:1,000) of anti-CFTR monoclonal antibody (mAb) 596 (University of North Carolina); CHIP antibody (anti-CHIP, catalog no. PA1-015; Thermo Scientific); anti-ubiquitin antibody (catalog no. SC: 8017; Santa Cruz Biotechnology). The blots were washed and CFTR proteins were visualized by enhanced chemiluminescence (Super Signal West Pico, chemiluminescent substrate, lot no. RE 227122; Thermo Scientific). The blots were stripped and probed with anti-β-actin antibodies (rabbit monoclonal IgM, 1:5,000, catalog no. 4970; Cell Signaling Technology) as a control for protein loading. Relative quantitation was performed by a densitometric analysis of band intensity using Quantity One software (Bio-Rad).