Immun blot lf pvdf membrane

HBEC6-KT and Calu-3 cells were lysed using RIPA Lysis Buffer containing Protease Inhibitor Cocktail powder (Sigma-Aldrich) for 60–90 min at 4 °C on a rocker. Lysates were then centrifuged at 16,000xg for 15 min with the supernatants collected for downstream immunoblots for ABCC4 and CFTR. Protein quantification was performed using a BCA protein assay. Twenty micrograms of protein were loaded on 4–15% gradient TGX Stain-Free Protein Gels and transferred to Immun-Blot LF PVDF membrane (Bio-Rad). The membranes were blocked with 1X TBS with 0.05% Tween 20 and 5% skim milk powder for 2 h at 25 °C. The membranes were then incubated with primary antibody ABCC4/MRP4 (1:40, Abcam, AB15602) or CFTR (1:5000, UNC-Chapel Hill, AB596) overnight. The membranes were then washed in 1X TBS with 0.05% Tween 20 and incubated with HRP-linked anti-rat secondary antibody (1:3000, Cell Signaling Technology, 7077S) or anti-mouse secondary antibody (1:3000, Cell Signaling Technology, 7076S) for 2 h at 25 °C. A chemiluminescence image of the blot was taken using the Bio-Rad Image Lab software. Full western blot images have been included and can be found in the supplementary information (Supplementary Fig. 4).

Twenty-four hours after transduction with one of the recombinant viruses—rMVA-k1, rMVA-k2, rMVA-k5, rMVA-M001, rMVA-hlHA or rMVA-NP+M1 (multiplicity of infection (MOI) was 2)—BHK-21 cells were collected, washed three times with PBS, resuspended in RIPA lysis buffer containing 1% NP-40 and lysed using ultrasonication. Cell debris was removed by centrifugation (15,000× g, 15 min, 4). A PageRuler Plus protein marker (Thermo Fisher Scientific Inc., Waltham, MA, USA) and the cell lysates were subjected to 12% SDS–PAGE (each sample contained 20 micrograms of protein). After electrophoresis under reducing conditions, the proteins were transferred to an Immun-Blot LF PVDF membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using wet electrophoretic transfer. The membranes were blocked in PBS containing 5% skim milk powder and 0.1% Tween 20 and then incubated overnight at +4 °C with antibodies against the 6x-His tag (HIS. H8-HRP, Invitrogen, Carlsbad, CA, USA) at a dilution of 1:20,000 or antibodies against beta-actin (AC-15-HRP, Abcam, Cambridge, UK) at a dilution of 1:50,000. Then, the membranes were washed 3 times in PBS with 0.1% Tween 20. Protein complexes were detected using Clarity Western ECL Substrate reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA) in accordance with the manufacturer’s recommendations and visualized using X-ray film (Fujifilm, Tokyo, Japan).