FIG. 2.

Coimmunoprecipitation of endogenous retromer subunits. (A and B) HeLa cells metabolically labeled with [³⁵S]methionine-cysteine were lysed with 0.5% (vol/vol) Triton X-100 and immunoprecipitated with rabbit anti-Vps26 (A) or rabbit anti-SNX1 (B). Washed immunoprecipitates were eluted by heating at 95°C for 5 min in the presence of 10 mM dithiothreitol and 1% (wt/vol) SDS and later diluted with lysis buffer. Samples were subjected to recapture with rabbit antibodies to Vps26, Vps29, Vps35, SNX1, SNX2, and the β3 subunit of the AP-3 complex as a negative control. Washed immunoprecipitates were analyzed by 12% acrylamide SDS-PAGE followed by fluorography. Two different exposure times are shown in panel A to better document the signal corresponding to Vps29 (this protein migrates as a doublet by SDS-PAGE). (C) HeLa cells were lysed with 0.5% (vol/vol) Triton X-100 and immunoprecipitated with (lane 3) or without (lane 2) a monoclonal antibody to SNX1. After being washed, the immunoprecipitates were eluted in sample buffer and analyzed by 4 to 20% acrylamide gradient SDS-PAGE and immunoblotting with a monoclonal antibody to SNX2. Ten percent of the input lysate was loaded in lane 1 as a control. (D) HeLa cells transfected with plasmids encoding Vps26-Myc (lane 1), CFP-SNX1 (lane 2), and HA-SNX2 (lane 3) were lysed and subjected to SDS-PAGE and immunoblotting with monoclonal antibodies to SNX1, SNX2, and HA and a polyclonal antibody to green fluorescent protein (GFP). The positions of molecular mass markers (in kDa) are indicated on the right in panels A to C. Abbreviations: IP, immunoprecipitation; Ab, antibody; IB, immunoblotting; Endo, endogenous; Ig-HC; immunoglobulin heavy chain; Ig-LC, immunoglobulin light chain.