Figure 4

KCC2 and pKCC2-S940 expression 24 h post-ischemia. (A & E) Representative KCC2 and pKCC2 expression at P7 and P10. The blots presented for each group were cropped from separate gels for improvement in clarity and conciseness of presentation. Vertical solid black lines delineate the separate blots between groups, while horizontal dotted black lines delineate separate proteins run on different gels for the same sample. All gels were run in the same experimental conditions (see methods for details). (Full-length blots presented in Supplementary Figure 4). (B & F) Line graphs represent KCC2 and pKCC2 expression in the left and right hemispheres at P7 and P10. (C & G) Boxplots represent mean expression of KCC2 and pKCC2 in ipsilateral hemisphere normalized to the contralateral at 24 h after ischemia at P7 and P10. P7 mice from the PB alone group showed ~30% downregulation of KCC2 expression similar to previous reports. The post-ischemic downregulation of KCC2 expression was rescued by of ANA12 + PB tested in a dose-dependent manner in this study. The rescue of post-ischemic downregulation of KCC2 was associated with a significant upregulation of pKCC2 at all three ANA12 + PB doses tested. Within-group comparison of ipsilateral and contralateral hemispheres was done using pair-wise t-tests; γP < 0.05, γγP < 0.01, γγγP < 0.001. Between-group comparison to PB alone was done using one-way ANOVA for P7, and independent t-tests for P10; *P < 0.05, **P < 0.01, ***P < 0.001. Bars with “#” denote significance between group comparisons to naïve control using one-way ANOVA for P7 and independent t-tests for P10; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001. Bars with “@” denote significance between groups compared to ANA12 5 + PB using one-way ANOVA for P7 and independent t-tests for P10; ^@P < 0.05, ^@@P < 0.01, ^@@@P < 0.001. (D & H) Bar graphs represent the ratio of pKCC2 to total KCC2 expression in the left and right hemispheres at 24 h post-ischemia P7 and P10. The ratio of normalized pKCC2 to normalized KCC2 was calculated for the same pup. (Between-group comparisons were done using one-way ANOVA; Post-hoc Bonferroni; *P < 0.05, **P < 0.01, ***P < 0.001 compared to PB-alone. Bars with “#” denote significance between group comparisons to Naïve Control; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001). For 1 h KCC2/pKCC2 WB data see Suppl. Figure 2.