Figure 3.

KCC2 is a calpain substrate. A, The KCC2 signal in an immunoblot decreased when rat brain homogenate was exposed to 31.5 U/ml recombinant rat calpain-2. An almost complete loss of the KCC2 immunosignal was observed after 30 min. The decrease in the KCC2 signal was blocked by the calpain inhibitor MDL-28170 (MDL). B, An immunoblot with an antibody directed against an N-terminal epitope of KCC2 (anti-NTD-KCC2) showed a truncated N-terminal fragment of KCC2 with a molecular weight (Mw) of ∼100 kDa. C, D, Ionomycin triggers a decrease in KCC2 protein level. C, Incubation of brain slices in the presence of the Ca²⁺ ionophore ionomycin (50 μm; 4 h) resulted in a clear reduction in KCC2 protein level, an effect that was blocked when ionomycin was applied together with MDL-28170. D, The mean values of the quantified immunoblot signals showed a significant decrease in KCC2 after ionomycin treatment (39.6 ± 2.6%; p < 0.05). The mean value of the ionomycin plus MDL-28170 samples did not differ from control (Ctrl; 86.6 ± 8.3%; p = 0.477). Statistical significance was assessed by One-way ANOVA with Scheffe's post hoc test. The values for n are given in the bar diagram. Error bars denote SEM.