CFTR Immunoprecipitation (IP) and Western Blot

Western blot was performed as described before.44 In brief, A549 cells (10 × 10e6) were collected after being treated with gRNA3-dCas9/VPR. Flasks were rinsed twice with PBS, and then PBS was removed. RIPA was added, cells were scraped, and lysate was placed in a microcentrifuge tube and centrifuged for 3 min at 13,000 × g. The supernatant was recovered and a protein assay was performed. Next, 500 μg of soluble lysate was placed in a microcentrifuge tube. 3 μL A2-596 antibody directed against CFTR NBD2 domain (obtained from Cystic Fibrosis Foundation) was added45, 46 and rotated overnight at 4°C. 50 μL of Pierce protein A/G magnetic beads was added and rotated overnight. Immunocomplexes were centrifuged and wash pellets were washed three times with 1 mL RIPA. RIPA was removed, sample buffer was added, and the solution was incubated at 90°C for 10 min. Samples were loaded onto a 6% gel for SDS-PAGE. Minigels were transferred to nitrocellulose. Blots were probed with anti-CFTR antibody 596 directed against CFTR NBD2 domain (1:1,000).