rp encodes a phosphorylated, conserved, widely expressed cytoplasmic protein. (A) Phosphorylation of wild-type RP. Whole cell extracts prepared from MNT-1 cells transiently expressing HA₃-RP or FLAG-RP were treated for 1 hour at 37°C in the presence (+)orabsence(–) of alkaline phosphatase. Samples were then subjected to immunoblotting analysis as indicated. The positions of molecular mass markers are indicated on the right. (B) The phosphorylation of RP was confirmed by metabolic labeling of HA₃-RP–expressing MNT-1 cells with 32P-orthophosphate in the presence (+) or absence (–) of alkaline phosphatase, followed by immunoprecipitation-recapture with antibodies to the myc (nonspecific control) or HA epitopes. The positions of molecular mass markers are indicated on the right. (C) Comparison of human and mouse RP amino acid sequence reveals an overall identity of 87% (shaded residues). The C>T transition converts amino acid 80 (underlined, glutamine) to a stop codon. (D) OriGene Technologies Northern blot showing expression of rp in mouse tissues. The blot was hybridized with the same 672-bp fragment described for Figure 2C. Molecular weight marker positions are indicated on the left. (E) rp message levels appear normal in kidney (left) and cultured melanocyte (right) mRNA. Note that a lower amount of rp/rp melanocyte mRNA was loaded, as judged by the intensity of the actin signal. Each lane contains 2 μg mRNA. (F-G) Immunofluorescence image of B6 (F) and rp/rp (G) melanocytes transfected with pHM6-RP and stained with anti-HA. No staining was evident in melanocytes transfected with vector alone (not shown). (H) Merged Nomarski and stained (affinity-purified chicken anti-RP peptide antibody recognizing amino acids 1-12 of the RP protein) image showing that RP-containing vesicles (green structures) and melanosomes (dark structures) do not colocalize extensively. (I-J) B6 (I) and rp/rp (J) melanocytes stained with affinity-purified chicken anti-RP peptide antibody. No staining was seen when the primary antibody (affinity-purified chicken anti-RP) was eliminated (not shown). Original magnification × 400 for panels F-J.