Figure 2: Hippocampal proteins phosphorylation and levels in Pyk2-deficient mice.
(a) Immunoblotting analysis of Pyk2, the related tyrosine kinase FAK, the active autophosphorylated form of Src-family kinases (pY-SFK, pTyr-420 in Fyn), Fyn and tubulin as a loading control in 3-month-old Pyk2+/+, Pyk2+/− and Pyk2−/− littermates. (b) Densitometry quantification of results as in a. Data were normalized to tubulin for each sample and expressed as percentage of wild type. (c) NMDA receptors subunits phosphorylated residues, total levels and PSD-95 were analysed by immunoblotting. (d) Results as in c were quantified and analysed as indicated in b. In b and d, statistical analysis was done with one-way ANOVA and Holm-Sidak’s multiple comparisons test or Kruskal–Wallis and Dunn’s test depending on the normality of distribution (see Supplementary Table 1 for tests used, values and number of mice). (e) PSD fraction was prepared from hippocampus of Pyk2+/+ and Pyk2−/− mice and NMDA receptor subunits and PSD-95 were analysed in this fraction by immunoblotting. (f) Quantification of immunoblots as in e. Data are expressed as a percentage of the mean values in wild-type PSDs. Two-tailed Mann and Whitney test (n=7+/+ and 5−/−): GluN1, t10=3.52, P=0.0056, GluN2A, t10=2.68, P=0.023, GluN2B, t10=2.69, P=0.022, PSD-95, t10=2.66, P=0.024. In a,c,e, molecular weight markers positions are indicated in kDa. In b,d, Holm-Sidak’s versus wild type, *P<0.05, **P<0.01, ***P<0.001 and ****P<10−4; significant differences between −/− and −/+ are indicated with °P<0.05, °°P<0.01 and °°°°P<10−4. In Dunn’s test (d) and Mann and Whitney’s test (f), significant differences versus wild type are indicated with # P<0.05, ### P<0.01 and #### P<10−4. In all graphs, data are means+s.e.m. Uncropped blots for a,c and e are shown in Supplementary Figs 5, 6 and 7, respectively.
