The immunoglobulin heavy chain of the IgM BCR is ubiquitinated. (A) MD4.B10.Br B cells (expressing an IgM^a BCR) were incubated with either no antibody, anti–IgM^a-btn, or anti–IgM^b-btn as indicated. Cells were then lysed and ubiquitinated proteins precipitated with p62 UBA–agarose. Ubiquitinated p62 UBA–binding proteins (P62 UBA) as well as whole-cell lysates (WCLs) were analyzed by reducing SDS-PAGE and Western blotting. Anti-IgM indicates goat anti–murine IgM plus rabbit anti–goat IgG–HRP (to detect BCR IgM heavy chain); No 1°, rabbit anti–goat IgG–HRP only (negative control); and SA-HRP, streptavidin-HRP (to detect biotinylated anti-BCR antibody). The anti-IgM and No 1° blots were treated identically except for the omission of the primary antibody. The molecular weights of the 2 bands in the WCLs are 70 and 80 kDa. The filled arrowhead indicates the 70-kDa BCR IgM heavy chain protein of the endogenous BCR detected in the p62 UBA precipitates. The open arrowhead indicates the position of the heavy chain of the anti–IgM^a-btn antibody. Shown are representative results from 1 of 3 independent experiments. (B) Splenocytes were lysed in RIPA buffer and the lysates precleared as indicated (αIgM + PGS indicates goat anti–murine IgM plus PGS; αIgG(H+L) + PGS, goat anti–murine IgG (H+L) plus PGS). The murine IgM BCR was then immunoprecipitated from the cleared lysates as indicated (+ indicates goat anti–murine IgM and PGS; –, PGS only). Immunoprecipitates were probed for the presence of ubiquitinated IgM by reducing SDS-PAGE and Western blotting with antiubiquitin mAbs (either the 6C1 or FK2, as indicated). The gray arrowheads below the blot indicate the lanes in which the IgM BCR was not precleared. The molecular masses of the 2 bands in the blots are 70 and 80 kDa. Shown are representative results from 1 of 3 independent experiments.