Fig 5.

Sex-specific developmental expression of NKCC1 and KCC2 protein. Immunoblots of KCC2 and NKCC1 with glyceraldehyde-3-phosphate dehydrogenase loading control for cortex (a), hippocampus (d) and thalamus (g) at ages P4 and P7 for both males and females (n=5 per group). (b) Quantification of western blot data for KCC2 and NKCC1 with individual data points and standard deviations displayed. Two-way analysis of variance of KCC2 levels showed differences in sex, age, and interaction. Sex: F(1,16)= 19.41 P=0.0004, age: F(1,16)=133.6 13 P<0.0001, interaction: F(1,16)=36.13 P<0.0001). Post hoc multiple comparison Sidak’s test showed a significant difference between KCC2 protein in males and females at P7 (P<0.0001). (c) Two-way analysis of variance of NKCC1 levels showed a difference by sex [F(1,16)=6.556 P=0.021] and interaction [F(1,16)=6.104 P=0.0251] but not age [F(1,16)=2.259 P=0.1523]. Sidak’s test showed a decrease in protein levels in females compared with males (P=0.0052). These results are consistent with a more mature expression pattern in females relative to males at P7. This pattern was not consistent across the hippocampus (d–f) which showed no difference in expression. KCC2 levels in the thalamus (g–i) were increased in females compared with males at P7 (Sidak’s P=0.039) similar to the cortex, but were dissimilar in that NKCC1 levels were no different at P7.