Fig. 5.

RBM3 interacts with IMP2. a Representative immunofluorescent staining of RBM3 and IMP2 in SVZ-NSPCs and SGZ-NSPCs from adult WT mouse brain. RBM3 (red), IMP2 (green) and DAPI (blue) were merged. Scale bar: 25 µm. b Representative immunofluorescent images from proximity ligation assay. SVZ-NSPCs and SGZ-NSPCs were challenged with OGD and reoxygenated for 3 h. WT NSPCs omitting primary antibodies (WT NC) or KO NSPCs served as negative controls (KO NC). Fluorescent dots indicating single RBM3-IMP2 interactions were counted in each cell, and 25 cells per group were used for quantification (n = 25). RBM3-IMP2 PLA signals (red) and DAPI (blue) were merged. Scale bar: 25 µm. Two-way ANOVA was used for statistical analysis; n.s. not significant; **p < 0.01; ***p < 0.001; ****p < 0.0001. All data are presented as mean ± SEM. c Representative image of proximity ligation assay of RBM3 and IMP2 in frozen brain sections from adult mice treated with HI and 7 days recovery. Scale bar: 50 µm. Contra contralateral (uninjured side), ipsi ipsilateral (injured side), LV lateral ventricle, cc corpus callosum, GCL granular cell layer, NC negative control without primary antibodies. d Schematic illustration of RBM3 and IMP2 constructs. Full-length (FL) RBM3 was fused with an N-terminal HA tag. Full-length (FL) IMP2 was truncated to N-terminal domain with two RNA-recognition motifs (RRMs) or C-terminal domain with four K Homology motifs (KHs); all of the constructs included an N-terminal FLAG tag. e, f Representative co-immunoprecipitation graphs of full-length or truncated RBM3 and IMP2 in HEK293 cells. Full-length FLAG-IMP2 was co-overexpressed with vector (Vec, negative control), or HA-RBM3 with or without RNase T1 pre-treatment. FLAG antibody was used to precipitate FLAG-IMP2. FLAG-IMP2 and HA-RBM3 in input or IP samples were detected by anti-FLAG or anti-HA antibodies using Western blot (e). A reciprocal CoIP was performed using full-length HA-RBM3 to precipitate full-length FLAG-IMP2, FLAG-RRMs or FLAG-KHs (f). Asterisks indicate target bands. HC heavy chain of IgG used for immunoprecipitation, LC light chain of IgG used for immunoprecipitation. g Representative Western blot of IMP2 expression in SVZ-NSPC and SGZ-NSPC after mock or OGD treatment in the presence or absence of RBM3