Fig. 5. Quantification of relative plasma membrane content of myc-TRPC4 and myc-TRL in HEK293 cells by surface biotinylation. (A) myc-TRPC4 or myc-TRL were transiently expressed in HEK293 cells together with YFP-EBP50. 48 hours after transfection, plasma membrane were biotinylated followed by cell lysis and incubation of lysates with avidin-agarose to recover biotinylated proteins. Total (left panel) and surface fraction (right panel) were separated by electrophoresis, electroblotted on PVDF and probed with an anti-myc antibody. Reactive bands were quantified with a densitometer. Optical densities of four dilutions of each sample were plotted against the amount of total (left panel) or biotinylated (right panel) proteins loaded. Lines indicate curves of best linear fit. The slopes of the correlations were determined. R_T (left panel) and R_S (right panel) were calculated by dividing S_WT, the slope obtained from the myc-TRPC4-expressing cells (filled circles, solid line) by S_TRL, the slope derived from the myc-TRL-expressing cells (opened circles, dotted line). R_T and R_S represent the relative total and surface contents, respectively of myc-TRPC4 and myc-TRL in transfected HEK293 cells. (B) The R_S/R_T ratio was determined from densitometry of three blot pairs similar to those presented in A and is shown as mean±s.e.m. (C) Degree of contamination with YFP (used as a marker of cytosolic proteins) or with non-biotinylated proteins. Monolayers were surface biotinylated (left panel) or exposed to the buffer alone (right panel) at 4°C for 30 minutes prior lysis. The cell extracts were incubated with avidin-agarose and the bound material, together with aliquots of the cell lysates, were separated by SDS-PAGE, transferred to PVDF and probed with either an anti-YFP (left panel) or an anti-myc (right panel) antibody. The absence of detectable avidin-bound YFP (lane 2), myc-TRPC4 (lane 5) or myc-TRL (lane 6) suggests no contamination by biotinylated cytosolic proteins or intracellular myc-tagged channels. T, cell lysate (30 µg of protein); B, avidin-bound (40 µl of precipitate). Results shown are representative of three independent experiments.

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