Fig. 8

Identification of tyrosine kinases that phosphorylate Nlg1. a, b In vitro kinase assay. Purified tyrosine kinases (GST-FGFR1, -TrkB, or -TrkC) were incubated in the presence of ATP with GST-Nlg1, GST-Nlg1-Y782A, or a positive substrate (NTRK), and run on a polyacrylamide gel. a pTyr immunoblot. Note the phosphorylation of NTRK (blue arrow) and GST-Nlg1 (red arrow) but not GST-Nlg1-Y782A by the three kinases. Note the autophosphorylation of the three kinases (green arrow). b Corresponding Coomassie gel. GST-Nlg1 shows several bands, corresponding to partial degradation. The strongest band at 40 kD contains the gephyrin-binding motif. The NTRK and kinases are present in low amounts and barely appear on the gel. c Protein extracts from COS cells expressing Nlg1-WT, Nlg1-Y782A, with or without FGFR1, TrkB, or TrkC, were immunoprecipitated with Nlg1 antibodies. Some cells were pretreated with FGFR1 or pan-Trk inhibitors. pTyr and Nlg1 immunoblots are shown. d pTyr signals normalized to Nlg1 levels in the different conditions (number of experiments within bars). e, f Protein extracts from cortical cultures at DIV 10–14 pretreated with the kinase inhibitors for 24 h were immunoprecipitated with Nlg1, Nlg2, or Nlg3 antibodies. pTyr and Nlg1/2/3 immunoblots are shown, respectively. g Average pTyr signals for the different inhibitors, normalized to the control, untreated condition (number of experiments within bars). h CA1 neurons from Nlg1 KO slices were electroporated with Nlg1-WT or Nlg1-Y782A, and AMPAR-mediated EPSCs were recorded upon stimulation of Schaffer’s collaterals, in comparison to unelectroporated neighboring neurons (black traces). Organotypic cultures were treated or not with 1 µM GNF5837 for 7 days before the recordings. i Average AMPAR-mediated EPSCs amplitude in the 3 conditions, normalized to non-electroporated controls (number of pairs indicated within the bars, from 3 independent experiments). Data in graphs d and g were compared by one-way ANOVA followed by Bonferroni post hoc test (ns: not significant, *P < 0.05, **P < 0.01, ****P < 0.001). Data in graph i were compared to the control condition by Wilcoxon matched-pairs signed rank test, and between themselves using one-way ANOVA followed by Tukey’s multiple comparison (ns: not significant, *P < 0.05, **P < 0.01). Data represent mean ± SEM