Figure 2. [³H]-TCA uptake activity and membrane expression of TM6 cysteine mutants.

[A] Uptake of [³H]-TCA was measured in COS-1 cells as described in “Experimental Procedures” and expressed as a percentage of the parental transporter, C270A. [B] Intact transfected COS-1 cells were treated with sulfo-NHS-SS-biotin as described in “Experimental Procedures” followed by Western Blot processing. Blots were probed with the anti-hASBT antibody (1:30,000 dilution) followed by horseradish peroxide-linked anti-rabbit immonuglobin (1:2000 dilution). Each blot was probed for the internal plasma membrane marker, α-integrin (150 kDa) and the absence of calnexin (90 kDa) (data not shown), an endoplasmic reticulum membrane protein representing the negative control in the biotinylated fractions. Marker lanes are shown on the left side of the individual blots. Mature glycosylated hASBT visualizes as the 41kDa band while the lower, 38 kDa band (not indicated) represents the unglycosylated species. [C] Densitometric analysis for cysteine mutants normalized to internal marker (α-integrin) and represented as a percentage of C270A parent. [D] [³H]-TCA uptake activity normalized to relative cell surface expression. Bars represent mean ± S.D. of three separate experiments with p ≤ *** 0.001, ** 0.01 and *0.05 respectively, using ANOVA with Dunnett’s post-hoc analysis.