Analysis of CFTR maturation and turnover by metabolic labelling.

<p>CFBE-wtCFTR cells were transfected with negative control or Cdc42 siRNA and cultured 48 h prior to pulse-chase experiments. Cells were pulse-labelled for 15 min with 100 μCi/mL of [³⁵S]methionine and [³⁵S]cysteine mix and then chased for 0, 0.5, 1, 2, and 4 h. CFTR was then immunoprecipited and subjected to SDS-PAGE. Bands corresponding to core-glycosylated (band B) and fully-glycosylated (band C) CFTR were quantified by densitometry for analysis. (A) Representative gels are shown. (B) Maturation of CFTR is evaluated as the ratio of band C detected at a given time relative to total CFTR (bands B+C). (C) CFTR turnover is displayed as the relative total CFTR amount (bands B+C) along the chase. Total CFTR amount is assigned a value of 100 in arbitrary units at the beginning of the chase (0 h), when the cells are transfected with negative control siRNA. (D) The rate of CFTR disappearance is estimated as the natural logarithm of the amount of CFTR (bands B+C) at a given time of chase relative to its amount at the beginning of the experiment (B₀+C₀). Displayed lines are the linear regressions to the data. Symbol and error bars are means ± SEM of the values at each point. The numbers of independent experiments used to build the graphs are indicated on the figures.