Biochemical characterization of the proB-cell surface complex on proB- and preB-cell lines. The proB-cell (JEA2, RS4.11), preB-cell (NALM6), and B-cell (Namalwa) lines were surface labeled with biotin and lysed with 1% NP-40 lysis buffer. Lysates (5 × 107 cells) were incubated with either IgG1 control, anti-VpreB (4G7), or anti-μ MoAbs. Immunoprecipitates were submitted to a gradient SDS-PAGE (5% to 15%) under reducing conditions and transferred onto immobilon P membrane. Cell surface biotinylated proteins were detected by streptavidin-peroxidase. The membrane was then successively incubated with the anti-VpreB 4G7 MoAb, which was shown by a peroxidase-conjugated goat antimouse IgG and finally with a peroxidase-conjugated mouse antihuman μ MoAb.
