RP interacts with pallidin, cappuccino, and muted. (A) The expression of pallidin was assessed in whole-cell extracts prepared from wild-type (C57BL/6J), reduced pigmentation (RP), and pallid (PA) mouse fibroblasts by immunoblotting using an antipallidin antibody. Immunoblotting with anti–α-tubulin provided a control for sample loading. The positions of molecular mass markers are indicated on the right. (B) RP coimmunoprecipitates with the BLOC-1 components pallidin, cappuccino, and muted. MNT-1 cells stably expressing HA₃-RP were metabolically labeled with [35S]methionine for 22 hours and extracted with lysis buffer containing 0.5% (wt/vol) Triton X-100. The extracts were then subjected to a first immunoprecipitation (1st IP) with mouse monoclonal anti-HA (lanes 5-8) or anti-Myc (lanes 1-4, controls). Washed immunoprecipitates were subsequently denatured by heating at 95°C for 5 minutes in the presence of SDS and dithiothreitol, diluted with lysis buffer, and subjected to a recapture immunoprecipitation step (2nd IP) using either rabbit antisera to pallidin (PA, lanes 1 and 5), cappuccino (CNO, lanes 2 and 6), or muted (MU, lanes 3 and 7) or mouse anti-HA antibody (lanes 4 and 8). The resulting immunoprecipitates were analyzed by SDS-PAGE on 4% to 20% gradient gels followed by fluorography. In all cases, the expected product was obtained indicating an association (*). The positions of molecular mass markers are indicated on the right. The slower migrating bands (arrows) likely represent nonspecific products because they are present in all immunoprecipitates including the controls.