Relative capacity of MoAb with high, intermediate, and low affinity for mIgM (Cμ2) to induce early protein tyrosine phosphorylation in Ramos B cells. Cells were incubated with high-affinity MoAb HB57, intermediate-affinity MoAb Mu53, low-affinity MoAb P24, or isotype control MOPC-21 at a concentration of 100 μg/mL (MoAb:cell ratio = 5 μg/105 cells) for 1 or 5 minutes before preparation of lysates and (A) analysis of lysates (20 μg protein) for tyrosine-phosphorylated proteins by SDS-PAGE, Western blotting with HRP-conjugated anti-P(tyr) Ab, and ECL, as described in Materials and Methods. A P(tyr)-positive control lysate (1:20 dilution) was run on both the 1- and 5-minutes lysate gels. The position of MW standards is shown on the left as approximate kilodaltons. The position and designation of several P(tyr) proteins that were densitometrically analyzed (as in Fig 6) is shown in the center as p137, p96, p78, and p60. (B) Blots were stripped and reanalyzed by Western blotting with rabbit anticatalase and HRP-conjugated goat antirabbit IgG and ECL, as described in Materials and Methods, as a control for the amount of loaded protein.
