Fig. 6. Turnover of CFTR glycosylation site variants with a single N-linked oligosaccharide. (A) Glycosylation site variant maturation and turnover as monitored by metabolic pulse-chase labeling. Cells were washed twice with methionine-free MEM and starved for 30 minutes in the same medium. [³⁵S]methionine was added to allow incorporation during a 20-minute pulse. Cells were then maintained in medium supplemented with 1 mM methionine, lysed with RIPA buffer at the times indicated, CFTR protein immunoprecipitated and the eluted proteins resolved on 7% polyacrylamide gels. (B) Effect of brefeldin A on turnover of glycosylation site variants. Wild-type and variant CFTR proteins were labeled in the presence of brefeldin A and pulse-chase experiments performed as described in A and Materials and Methods. (C) The amount of labeled protein remaining after the indicated chase periods was quantified by electronic autoradiography image analysis and is shown as a percentage of the initial label.