Fig 2: Pyk2 enhances tau pathology in vivo. A) The murine Thy1.2 promoter drives human P301L tau and FLAG-tagged Pyk2 expression in pR5 and Pyk2 transgenic mice, respectively. Representative immunofluorescence staining of brain sections from 12–15-month-old pR5 and double transgenic pR5/Pyk2 animals shown in (B) with magnified view in (C). Scale bars represent 500μm (B) and 50μm (C). D) Quantification of the pY18-tau positive area in the hippocampus of transgenic brains. Mean±s.e.m, n = 7 mice per group, two-tailed t test, **p < 0.01. E) Validation of pY18-tau antibody using immunoblots of a lysate from human tau-expressing HEK cells. WT, wild-type; Y18A, tyrosine to alanine mutant. F) Representative immunoblots of sequentially (RAB/RIPA) fractionated hippocampal homogenates from 12–15-month-old pR5 and pR5/Pyk2 mice. Both pY18-tau and HT7 antibodies react with transgenic human tau but not endogenous mouse tau. ‘h’ indicates human transgenic tau, and ‘m’ indicates endogenous murine tau. G) Quantification of immunoblots in (F). Fold changes are relative to the pR5 group. Mean±s.e.m, n = 4–6 mice per group, two-tailed t test, #p = 0.075, *p < 0.05.

Fig 3: Pyk2 phosphorylates tau in vitro. A) Coomassie staining and immunoblots of purified recombinant His-tagged Tau-V5 and Pyk2-V5 proteins. The full-length forms of the recombinant proteins are indicated at the expected molecular weight. Carboxy-terminally cleaved Pyk2 was detected using both anti-V5 and anti-C-terminal Pyk2 antibodies. N-terminal and C-terminal Pyk2 antibodies were raised against an amino- and carboxy-terminal epitope of human Pyk2, respectively. B) Immunoblots of pY18-tau after incubation of recombinant tau with or without recombinant Pyk2 in the in vitro phosphorylation reaction. C) Immunoblots of pY18-tau after incubation of recombinant tau and Pyk2 with or without ATP in the in vitro phosphorylation reaction. D) Quantification of immunoblots in (C). Mean±s.e.m, n = 3 per group, two-tailed t test, ***p < 0.001. E) Representative immunoblots of a time course assay of Pyk2-catalyzed tau phosphorylation in vitro. Reactions were carried out at 30°C in the presence of 0.5μM recombinant Pyk2 and 1.9μM recombinant tau. F) Normalized pY18-tau levels in (E) were plotted as a function of incubation time, with fold-changes relative to time point ‘0 s’. The curve was generated by nonlinear regression. Three independent experiments were performed.

Fig 4: Comparison of the hypoxia effect on pPyk2 levels and MMP‐9/MMP‐2 activation in control (Lrp1 +/+) and LRP1‐deficient fibroblasts (Lrp1 −/−). Quiescent Lrp1 +/+ and Lrp1 −/− fibroblasts were exposed to normoxic or hypoxic conditions for 18 hrs. (A) Representative Western blot analysis showing LRP1, pPyk2, total Pyk2 and β‐tubulin levels and bar graphs showing the mean ± S.D. of LRP1 levels normalized to β‐tubulin (B) and pPyk2/total Pyk2 ratio (C). (D) Representative zymography analysis showing MMP‐9 and MMP‐2 activity levels and bar graphs showing the mean ± S.D. of MMP‐9 (E) and MMP‐2 activity levels (F). Results are shown as the mean ± S.E.M. of three independent experiments performed in triplicate. ***P < 0.001 versus normoxia. (G) Representative scheme showing the crucial role of LRP1 up‐regulation in hypoxic cardiac fibroblasts in cardiac remodelling.

Fig 5: Pyk2 is implicated in acute cocaine responses. (A), Immunoblot for phospho-tyrosine and total Pyk2 after Pyk2 immunoprecipitation (IP) from striatal extracts of wild-type mice killed 10 min after i.p. injection of saline (Sal) or cocaine (20 mg/kg, Coc). (B), Densitometry quantification of results as in A. Data are ratios of phospho/total Pyk2 for each sample and expressed as percentage of the mean ratios in saline-treated samples (n = 7 mice per group). (C), Locomotor activity of Pyk2+/+ (n = 10 mice) and Pyk2-/- mice (n = 9) after a first cocaine injection (20 mg/kg, Coc, arrow) 30 min after mice were placed in the open field. (D), The same mice received a second injection of cocaine in the same conditions as in C, 13 days later. (E), Pairwise comparison of the distance traveled 0–15 minutes following the first and the second injection in Pyk2+/+ and Pyk2−/− mice (from data in C and D). (F), Sensitization ratios calculated as the distance traveled 0–15 min after the second injection (day 14) divided by the distance traveled during the same period after the first (day 1) cocaine injection (from data in E), in Pyk2+/+ and Pyk2−/− mice. (B-F), Data are means ± SEM, indicated by a shaded area in C and D, and horizontal bars in B, E and F. t-test in B and F, 2-way ANOVA followed by post-hoc Sidak’s multiple comparisons test in C-E. ns, not significant, *p < 0.05, **p < 0.01. See Supplementary Table 1 for detailed statistical analyses and Supplementary Figure 3 for full length blots.