Western Blot Analysis of Cultured CFs

Total protein was extracted from the cultured CFs as previously described (38 (link),39 (link)). Briefly, the lysates were collected by centrifugation at 10,000 × g. The protein concentrations were determined using a BCA protein assay kit. Fifty micrograms of protein extract were used for SDS-PAGE. The proteins were then transferred onto nitrocellulose membranes, blocked with 5% skimmed milk powder and then probed with various antibodies overnight at 4°C. Following incubation with a secondary peroxidase-conjugated antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA; at a 1:10,000 dilution), signals were visualized with FluorChem E (Cell Biosciences, Santa Clara, CA, USA). Specific protein expression levels were normalized to GAPDH for total cell lysates and cytosolic proteins on the same nitrocellulose membrane.