Sedimentation velocity analysis of HA₃-RP. Cytosol prepared from MNT-1 cells stably expressing HA₃-RP was fractionated by ultracentrifugation on a 2% to 15% (wt/vol) linear sucrose gradient. (A) Samples representing 3.3% of the fraction volume were analyzed for the presence of HA₃-RP by immunoblotting (IB) using monoclonal anti-HA antibody. (B) The presence of the HA₃-RP - pallidin complex was assessed on samples (∼ 40% of the volume of each fraction) by immunoprecipitation using antibody to pallidin and immunoblotting using anti-HA antibody. Note that RP and pallidin cosediment in fractions 6 to 8 and that the mobility of RP is shifted in these fractions. (C) The sedimentation of endogenously expressed pallidin was analyzed by immunoblotting using antibodies to pallidin. Note that HA₃-RP and pallidin cosediment in fractions 6 to 8 with a peak in fraction 7. (D) The sedimentation of endogenously expressed HPS4 was assessed by immunoblotting; HPS4 peaked in fraction 8. (E) Samples from fractions 2, 4, 5, and 7 representing 2.5%, 0.8%, 2.5%, and 5.8% of the fraction volume, respectively, were analyzed by IB using anti-HA antibody. Notice that the upper band of HA₃-RP (phosphorylated form) is predominant in fraction 7. The positions of standard proteins (sedimentation coefficients given in Svedberg units) in the gradient are indicated on the top. The positions of molecular mass markers are indicated on the left.