Figure 7: G3BP1 overexpression blocks the Gag–eEF2 interaction.

(a,b) HEK293T cells were co-transfected with mCh-Gag, Flag-eEF2 and G3BP1-GFP in different concentrations. After 48 h, cells were collected for IP analysis with anti-RFP followed by immunoblot analysis and at different time points (c). Data shown are representative of two independent experiments. (d) Schematic representation of the experimental protocol, including the timing of live cell microscopy (LCM). HeLa cells were co-transfected with G3BP1-GFP and mCh-Gag (e,h) or G3BP1-GFP and mCh-GagQ7A/Q9A (f) and observed under LCM beginning at 20 h post transfection at 15 min intervals. Scale bar, 10 μM (see also Supplementary Movies 1 and 2). (g) Cells from e,f were subjected to PLA. Spots per cell showed the relative quantitation of Gag–G3BP1 (endogenous) association. Data are represented as mean±s.e.m. of three independent experiments. Statistical analysis was performed with a one-way ANOVA combined with the Bonferroni’s post test. (i) Insets represent high-resolution magnification of cell in h where SGs disappeared over time of the experiment. Scale bar, 5 μM (see also Supplementary Movie 3). (j) Z-stacks were acquired during LCM and imported into IMARIS software. 3D reconstruction was utilized to calculate G3BP1 granule size (volume—grey line). Quantification of SG is shown (#SG—black line). Each data point is the mean collected from four cells analysed.