A, Western blot analysis of cell lysates prepared from ARPE19 cells transiently transfected with the wild-type (WT) or the mutant forms of hTPPT (FLAG - tagged everywhere) was performed using monoclonal anti-FLAG antibody (upper panel) or monoclonal anti-β-actin antibody (lower panel). The images are representatives of two independent experiments with similar results. B, Effect of mutations in N-linked glycosylation sites of hTPPT on carrier-mediated TPP uptake. ARPE19 cells transiently expressing mutants (or control, wild-type hTPPT, WT) were used for TPP uptake measurements. [3H]-TPP (0.3 μM) was added to the incubation medium (KR buffer of pH 7.4) at the onset of incubation, and uptake was measured after 5min of incubation. Carrier-mediated TPP uptake by the induced system was calculated as described in “Materials and Methods”. Data are mean ± S.E. of six to eight separate uptake determinations. *p < 0.01.