Fig 2: Detection of NR2B protein in the cortex by immunofluorescence. (A) Representative samples (magnification, ×200) from each group (n=6). Green staining indicates NR2B expression in the cells. (B) Ratio of NR2B-positive cells in each group. Data are presented as the mean ± standard deviation. *P<0.05 vs. sham group. NR2B, Glutamate receptor ionotropic, N-methyl-D-aspartate 2B.

Fig 3: Activated NR2B (Tyr1472) expression, the enhanced interaction of NR2B with Src, and changes in the levels of p-Src and p-NR2B after the administration of 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4-d] pyrimidine (PP2) to the immature rat HI brain injury model. (A) Immunoblots showing the levels of multiple proteins. Primary antibodies are listed on the left. (B) Quantification of NR2B (Tyr1472) (p-NR2B) and NR2B levels at 2 h post injury (HI 2 h). N = 7 animals per group. (C) Coimmunoprecipitation was conducted using right hemisphere homogenates with the Src antibody or rabbit IgG as a control. Immune complexes were detected using immunoblotting (IB) with anti-Src and anti-NR2B antibodies. (D) Immunoblots showing the levels of multiple proteins. Primary antibodies are listed on the left. (E) Quantification of the levels of p-Src and Src at HI 2 h. N = 4–6 animals per group. (F) Quantification of the levels of p-NR2B and NR2B at HI 2 h. N = 4–6 animals per group. *p < 0.05 and **p < 0.01.

Fig 4: Overexpression of CK2 alleviated brain injury in ICH rats. pcDNA-CK2 and pcDNA-NC were transfected into ICH rats. (A) Expression of CK2 was detected using Western blot (n = 6). (B) Neurologic score was determined by the Forelimb placing test, corner turn test and Bederson score. (C) Analysis of brain edema. (D) Expression of cleaved caspase3, NR2B, NR2B phosphorylation sites S1480, S1303, and Tyr1472 were determined in rats using Western blot (n = 6). (E) Representative gel images and quantification of co-immunoprecipitation (co-IP) show the level of NR2B-PSD95 complex in rat brain tissues (n = 3). Data were expressed as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 5: Increased excitability of PV+ interneurons in Htr3a-/- mice by NMDAR upregulation. (A) Significant increase in GluN2B immunoreactivity (green) in the PV+ (red) neurons in the CA1 region of Htr3a-/- mice (bottom row), compared with controls (top row). Scale bar: 50 µm. (B) Quantified data showing a significant increase of GluN2B in PV+ neurons in the CA1 region of Htr3a-/- mice. (Student's t test, p = 0.0005, n = 93 cells from 3 WT mice, n = 100 cells from 3 KO mice). (C) Representative traces of evoked AMPAR- (held at -70 mV) and NMDAR-current (held at +40 mV) in PV+ neurons in the CA1 region of Htr3a-/- and WT mice. Scale bar: 20 pA, 100 ms. (D) Normal AMPAR current but increased NMDAR current in PV+ neurons in the CA1 region of Htr3a-/- mice. (For AMPAR current, two-way ANOVA, F1, 140 = 1.831, p = 0.0001; for NMDAR current, two-way ANOVA, F1, 140 = 31.27, p < 0.0001. n = 13 cells from 3 WT mice, n = 9 cells from 3 KO mice). (E) Voltage traces evoked by 300 pA current injection before and after D-APV (50 µM) treatment of Htr3a-/- and WT mice. Scale bars represent 100 ms, 20 Mv. (F) Firing number plotted against depolarizing currents. (n = 9 cells from 3 WT mice, n = 12 cells from 3 KO mice; two-way ANOVA, WT vs. KO, F1,171= 25.95, p < 0.0001; two-way ANOVA by matching factors, WT vs. WT APV, F1,8= 9.451, p = 0.0152; two-way ANOVA by matching factors, KO vs. KO APV, F1,11= 10.60, p = 0.0077; two-way ANOVA, WT vs. KO APV, F1,19= 0.3283, p = 0.5734). Data are presented as boxplots (median and 5th-95th percentile whiskers), or as mean ± SEM., * p < 0.05; ** p < 0.01, *** p < 0.001, **** p < 0.0001.