*4.3. Pulse-Chase and Immunoprecipitation*

BHK cells lines stably expressing CFTR variants were starved for 30 min in methionine-free α-modified Eagle's medium or minimal essential medium and then pulsed for 30 min in the same medium supplemented with 100 µCi/mL [35S] methionine. After chasing for 0, 0.5, 1, 1.5, 2, and 3 h in a-modified Eagle's medium with 8% (*v*/*v*) fetal bovine serum and 1mM non-radioactive methionine, cells were lysed in 1 mL of Radioimmunoprecipitation assay (RIPA) buffer [1% (*w*/*v*) deoxycholic acid, 1% (*v*/*v*) Triton X-100, 0.1% (*w*/*v*) Sodium dodecyl sulfate (SDS), 50 mM Tris, pH 7.4, and 150 mM NaCl]. The immunoprecipitation (IP) was carried out using the anti-CFTR 596 antibody in independent experiments and Protein G–agarose or Protein A–Sepharose beads. Immunoprecipitated proteins were

eluted from the beads with sample buffer for 1h at room temperature and then electrophoretically separated on 7% (*w*/*v*) polyacrylamide gels. Gels were pre-fixed in methanol/acetic acid (30:10, *v*/*v*), washed in water and, for fluorography, soaked in 1M sodium salicylate for 60 min. After drying at 80 ◦C for 2 h, gels were exposed to X-ray films and further analyzed and quantified by densitometry.
