Immunoprecipitation and immunoblotting were performed as described previously. CH27-HA-FcRL1-YFP cells (1 × 107 cells/ml) were preincubated with serum-free RPMI 1640 at 37°C for 30 min (starvation) and resuspended at 5 × 107 cells/ml in fresh serum-free medium. Cells were stimulated with F(ab′)2 fragment anti-mouse IgM (10 μg/ml) or biotin-conjugated F(ab′)2 fragment anti-HA (15 μg/ml) at 37°C. Cells were lysed with 1% Triton X-100 lysis buffer. For the immunoprecipitation assay with GSH beads, cell lysates were precleared with GSH beads link-coupled with GST protein for 2 hours. Then, the lysed cells were immunoprecipitated with GSH beads link-coupled with GST or GST-SH2 at 4°C for 6 hours. During the detection of the interaction between c-Abl–mCherry-GST and FcRL1, the precleared step was omitted. To quantify the immunoprecipitated proteins, immunoblots were probed with rabbit anti-YFP antibody. For the immunoprecipitation assay with anti-HA, the HA-FcRL1-YFP chimeric receptors were captured by biotin-conjugated F(ab′)2 fragments anti-HA and then precipitated by beads conjugated with streptavidin.

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