Fig. 3.

BCR induced JNK activation is required for efficient receptor trafficking to late endosomes. (A) Splenic B cells from WT and 3H9/Vκ8 mice were assayed for the induction of MAPK activation after BCR stimulation with anti-IgG/M F(ab′)₂ antibodies, and total cell lysates probed in Western blottings with the phosphorylated and total pools of the indicated MAPKs. The normalized ratio of the density for each phospho-MAPK band to the corresponding total MAPK band is provided. (B) Purified B cells were preincubated for 30 min with inhibitors to p38 (SB202190, 0.3 μM; Calbiochem), JNK (SP600125, 10 μM; Invitrogen), or ERK1/2 (25 μM; Calbiochem). Indicated samples were incubated for 18 h with JNK Inhibitor III (100 μM; Calbiochem). Aliquots were subsequently stimulated with FITC-conjugated anti-IgG/M (H+L) F(ab′)₂ antibodies (green) for 30 min. Samples were then fixed, counterstained with ID4B (red), and visualized by confocal microscopy. (C) WT splenic B cells were cultured in the presence of a pan-JNK inhibitor (10 μM, SP600125) for 30 min, and then stimulated with FITC-conjugated anti-BCR antibodies and PMA for 30 min. Samples were then fixed, stained with ID4B, and visualized by confocal microscopy. Results are representative of 3 independent experiments. (D) Quantitation of results provided in C performed as described in Fig. 1.