Increased TYRP1 cycling through the plasma membrane in BLOC-2⁻ melanocytes. (a) Comparison of cell surface levels of TYRP1, TfR, or PMEL among BLOC-2⁻, BLOC-2^−C, and BLOC-2^R melanocytes by flow cytometry after labeling of whole cells on ice with unlabeled primary and AF488-conjugated secondary antibody. The mean fluorescence intensity (MFI) ± SD over 4–15 experiments, each performed in duplicate or quadruplicate, was normalized to 1.0 for BLOC-2^R melanocytes within each experiment. (b–d) Quantification of TYRP1 (b), PMEL (c), and TfR (d) endocytosis rates. BLOC-2⁻, BLOC-2^−C, or BLOC-2^R cells were incubated with unlabeled antibodies on ice and loss of surface antibody (detected with fluorescent secondary antibody) over time at 37°C was quantified by flow cytometry. MFI at each time point was calculated and plotted relative to MFI at time 0 (defined as 100%). Values (means ± SD) were from two to eight separate experiments performed in duplicate. (b and d) P-values are indicated for comparison of BLOC-2^R to BLOC-2⁻ cells. Values in c were not significantly different. (e and f) Quantification of TYRP1 flux through the plasma membrane. Uptake of saturating levels of fluorescent anti-TYRP1 antibody by BLOC-2⁻, BLOC-2^R, or BLOC-1–deficient melan-mu cells (BLOC-1⁻) at 37°C was quantified over time by flow cytometry. In f, cells were left untreated or were treated with 10 µg/ml brefeldin A (BFA) for 1 h before and during the experiment as indicated. Values (means ± SD) were from at least three independent experiments performed either in duplicate or quadruplicate and represent raw MFI (e) or a normalized value (f) calculated as the percentage of the MFI value for untreated BLOC-2⁻ cells at 2 h. P-values shown in e correspond to all points in the graph except the initial time point; p-values in f are shown only for differences deemed significant between −BFA and +BFA samples. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant.