Figure 3: DSBs generated by antibody class switch recombination induce inactivation of GSK3β by p38 MAPK.

(a) Fresh B cells (−) and B cells activated with LPS and IL-4 for 24 and 48 h were examined by western blotting for P-S³⁸⁹ GSK3β, total GSK3β and γH2AX. (b) GSK3β activity in B cells activated with LPS and IL-4 for 24 and 48 h were examined by in vitro kinase assays (n=3, ±s.e.m.). *P<0.05 as determined by t-test. (c) B cells from WT and AID KO mice were stimulated for 48 h and the levels of P-S³⁸⁹ GSK3β, P-S⁹ GSK3β, total GSK3β and γH2AX were determined by western blot analysis. (d) Western blot analysis for P-S³⁸⁹ GSK3β and total GSK3β using nuclear and cytosolic extracts from B cells activated for 24 and 48 h. GAPDH and histone are shown as controls. (e) B cells activated with LPS and IL-4 for 48 h were examined by immunostaining and confocal microscopy for the presence of γH2AX (green), P-S³⁸⁹ GSK3β (red) and TOPRO nuclear stain (blue). Scale bar, 3 μm. Data are representative of three or more independent experiments.