Fig. 4: MAPK4 KO mice exhibit enhanced proximal BCR signaling and B cell activation.

A, B Splenic B cells from WT and MAPK4 KO mice were incubated with sAg at different time points, then fixed, permeabilized, and stained for pBTK. Cell images (A) were captured using a Leica confocal microscope, and Pearson’s correlation coefficients (B) were calculated to evaluate the colocalization of pBTK with the BCR. Scale bar: 2.5 μm. C Phospho-flow cytometry analysis of the MFI of pBTK in B cells from WT and MAPK4 KO mice stimulated in vitro with sAg at the indicated time points. D Western blot analysis of the pBTK, BTK, pSYK, SYK, pCD19 and CD19 expression levels in B cells from WT and MAPK4 KO mice stimulated in vitro with sAg for 0, 5, 10, and 30 min. E Splenic B cells from WT and MAPK4 KO mice were incubated with sAg at different time points, then fixed, permeabilized, and stained for pY. Cell images were captured using a Leica confocal microscope. Scale bar: 2.5 μm. F Colocalization coefficients of pY and BCR were compared between WT and MAPK4 KO mice. G Phospho-flow cytometry analysis the MFI of pY in B cells from WT and MAPK4 KO mice stimulated in vitro with sAg at the indicated time points. H Western blot analysis of pY expression levels in B cells from WT and MAPK4 KO mice stimulated in vitro with sAg for 0, 5, 10, and 30 min. I, J Flow cytometry analysis the MFI of BCR (I) and ACTIN (J) in B cells from WT and MAPK4 KO mice stimulated in vitro with sAg at the indicated time points. K Flow cytometric analysis of BCR internalization within B cells from WT and MAPK4 KO mice following stimulation with sAg (n = 4). L Flow cytometry analysis of intracellular calcium levels in B cells from WT and MAPK4 KO mice. M, N Proportion of CD86⁺CD69⁺B220⁺ B cells (M) and comparison (N) in B cells from WT and MAPK4 KO mice after 24 h of stimulation with sAg (n = 5). O, P Flow cytometric analysis was performed to compare the expression of CD69 (MFI) in B cells from WT and MAPK4 KO mice after 24 h of stimulation with sAg (n = 3). Q, R Splenic B cells from WT and MAPK4 KO mice were incubated with sAg at different time points, then fixed, permeabilized, and stained for pWASP. Cell images (Q) were captured using a Leica confocal microscope, and Pearson’s correlation coefficients (R) were calculated to evaluate the colocalization of pWASP with the BCR. Scale bar: 2.5 μm. S Phospho-flow cytometry analysis the MFI of pWASP in B cells from WT and MAPK4 KO mice stimulated in vitro with sAg at the indicated time points. T Western blot analysis of pWASP expression levels in B cells from WT and MAPK4 KO mice stimulated in vitro with sAg for 0, 5, 10, and 30 min. Representative results from three independent experiments are presented for all of the above. *P < 0.05; **P < 0.01; ***P < 0.001.