Fig. 6: NMDAR signaling stabilizes RBM4 and promotes its nuclear translocation.

A GCs were treated with different concentrations of NMDA as in Fig. 5A. Immunoblotting of RBM4 protein and RT-PCR of Rbm4a/b mRNA were performed. Folds of RBM4 induction (normalized to GAPDH) were indicated below the blot (n = 3). B GCs were sequentially treated with AP5 or CK59 and NMDA as in Fig. 5C, E. Immunoblotting of RBM4 and GAPDH was performed. Induction folds were indicated as in (A). C Schematic diagram shows the S309A mutant of RBM4. GCs were transiently transfected with the vector expressing FLAG-RBM4 (wild-type or SA) followed by mock or NMDA treatment. Immunoblotting and fold changes were as in (A). D GCs were mock or NMDA treated alone or sequentially treated with AP5 or CK59 and NMDA. Immunofluorescence was performed using anti-RBM4. Cells were counterstained with DAPI. The scale bar represents 25 μm. Insets show enlarged images of a representative cell. Bar graph shows the relative ratio of RBM4 in the nucleus (N) vs. the whole cell (T) (average ± SEM, ***P < 0.001). For each group, ~60 cells were measured. Mock was set to 1. E Subcellular fractionation was performed in mock or NMDA-treated GCs. Immunoblotting was performed using antibodies against RBM4, Lamin A/C, and GAPDH. F GCs were transfected with the vector of FLAG-RBM4 (WT or SA) followed by mock or NMDA treatment. Immunofluorescence was performed using anti-FLAG. No signal was detected in untransfected cells (top panels), indicating the specificity of anti-FLAG. Scale bar, inset, and bar graph were as in (D). For the bar graph, mock-treated RBM4-transfection was set to 1. G Rbm4dKO GCs were mock-transfected or transfected with the FLAG-RBM4 (WT or SA) vector. RT-PCR and immunoblotting of indicated RNA/protein were performed. Relative Bdnf expression levels were indicated (n = 3); WT was set to 1.