Figure 4.

Tyro3 activation drives membrane insertion of GluA2. (A) Representative images of GluA2 surface expression, detected by 30 min incubation with a GluA2 antibody (Blanco-Suarez et al., 2018), in cortical neurons prepared from WT versus Tyro3^−/− mice, at 18DIV. (B) Imaris quantification of GluA2 surface expression per cell volume (V_CT, quantified by CellTracker Red staining), normalized to WT, in WT versus Tyro3^−/− neurons. In this panel, and in panels (C,E,G), n is the total of number of neurons analyzed from 3 to 5 separate culturing experiments (of the indicated genotypes) for each of the various experiments, with the number of culturing experiments indicated by the number of plotted data points. The value of these plotted points is the average of all cells analyzed in the same culturing experiment. (C) Imaris quantification of cell volume (V_CT) normalized to WT in WT versus Tyro3^−/− neurons. (D) Representative images of GluA2 surface expression in cortical neurons at 18DIV treated for 7 days with vehicle control (TBS, left), 20 nM recombinant Gas6 (middle), or 20 nM recombinant Gla-less Gas6, which binds but does not activate Tyro3 (right). (E) Imaris quantification of GluA2 surface expression per cell volume (V_CT) in neurons incubated with the indicated reagents for the indicated times, normalized to TBS. (F) Representative images of GluA2 surface expression in cortical neurons treated for 1 day with vehicle control (TBS, left), 20 nM recombinant mouse Gas6 (middle), or 20 nM recombinant mouse Gas6 plus 100 nM Annexin V (right). (G) Imaris quantification of GluA2 surface expression per cell volume (V_CT) in neurons incubated with the indicated reagents for 24 h, normalized to TBS. p-values: * <0.05, ** <0.01. Scale bars: 20 μm.