CNO, pallidin, and muted interact. (A-C) Fibroblasts from C57BL/6 (A), pallid (B), and cappuccino (C) fibroblasts were metabolically labeled with ³⁵S-methionine and subjected to immunoprecipitation-recapture assays using preimmune serum (PI) or the combination of antibodies indicated at the top of each panel. In this technique, tissue extracts are subjected to immunoprecipitation under nondenaturing conditions. Antibody-protein complexes are recovered using protein A Sepharose, denatured, and immunoprecipitated again using the same or a different antibody. Relative to C57BL/6 fibroblasts, CNO and muted (MU) are decreased in pallid fibroblasts, as are pallidin (PA) and muted (MU) in cappuccino fibroblasts, suggesting that these proteins interact. The mutant form of CNO (CNO*) is only detectable in cappuccino fibroblasts, as expected. (D) Immunoprecipitation-recapture assays in H4 cells reveal that pretreatment with CNO antibodies precipitates PA and pretreatment with PA antibodies precipitates CNO, indicting an interaction between CNO and PA. The doublet seen with anti-PA is characteristic of H4 cells.²⁰  Its origin is unknown, but may represent proteolysis during handling. (E) Pretreatment with CNO antibodies precipitates MU in H4 cells, indicating an interaction between and CNO and MU. (F) The mutant form of CNO (CNO*) is unable to associate with pallidin in fibroblasts, indicating that failure to produce an intact BLOC-1 complex underlies the HPS phenotype in cno/cno mice. (G) CNO does not interact with the σ3 subunit of the AP3 complex in H4 cells. The migration of molecular weight markers (kDa) is shown to the left of each panel.