FIGURE 9.

Active TAT-STEP protein blocks phosphorylation and translocation of Pyk2 to PSD upon KCl depolarization. A, WT or STEP KO mouse brain slices were pretreated with TAT-STEP WT (active), TAT-STEP C/S (inactive), or active STEP without TAT for 30 min; stimulated with 40 mm KCl for 2 min; and frozen on dry ice before isolation of PSD fractions. Phosphorylation levels were probed with phosphospecific antibodies (Tyr(P)⁴⁰² Pyk2 or Tyr(P)²⁰⁴ ERK1/2) and normalized to total Pyk2 and ERK1/2 levels, respectively, and then to β-actin as a loading control. Error bars indicate the SEM of at least three independent experiments (in this and subsequent panels). All values were compared with those in WT aCSF samples (**, p < 0.01; one-way ANOVA with post hoc Tukey test; n = 4). B, primary hippocampal neurons were pretreated with active TAT-STEP WT or inactive TAT-STEP C/S followed by KCl (40 mm; 2 min) stimulations. Colocalization of Pyk2 and PSD-95 was visualized using immunostaining with anti-Pyk2 (red) and anti-PSD-95 (green) antibodies. Arrowheads in the merged images indicate colocalized puncta. The number of Pyk2/PSD-95-colocalized puncta was counted per 10 μm of dendrites; 17 neurons were used for quantification per treatment. All values were compared with control (**, p < 0.01; one-way ANOVA with post hoc Tukey test; n = 17). pERK1/2, phospho-ERK1/2, pPyk2, phospho-Pyk2.