FIGURE 3.

Membrane assembly of pre-BCR requires μHC core fucosylation. A, cell surface biotinylation and immunoprecipitation of pre-BCR from 70Z/3, 70Z/3-KD, and 70Z/3-KD-re cells. The surface cellular proteins of the indicated cells were biotinylated before cell lysis. Whole lysates were immunoprecipitated (IP) with anti-pre-BCR antibody. The samples were subjected to 10% SDS-PAGE. After Western blotting (WB), the membrane was incubated with streptavidin-HRP. Quantification of expression levels of pre-BCR was analyzed by NIH Image 1.63. B, core defucosylation of μHC in 70Z/3-KD cells. The cell lysates were immunoprecipitated by anti-μHC Ab, and the immunoprecipitates were resolved by SDS-PAGE on 7.5% gel, transferred to a PVDF membrane, and probed with the anti-μHC Ab (upper panel) and AOL (lower panel). Abrogation of the core fucose of μHC was detected by AOL staining. C, impaired interaction between μHC and λ5 in 70Z/3-KD cells. Cell lysates were immunoprecipitated with anti-μHC Ab and analyzed by Western blot with antibody to λ5 (upper panel) and cell lysates were immunoprecipitated with anti-λ5 Ab and analyzed by Western blot with antibody to λ5 (lower panel). D, molecular interactions between λ5 and μHC, detected by an optical biosensor (n = 3). The μHCs were extracted from the lysates of 70Z/3, 70Z/3-KD, and 70Z/3-KD-re cells. Monobiotinylated λ5 were coated. The data reflect the proportion of μHC associated with λ5.