Fig. 2

Biochemical characterization of the RASopathy-causing SPRED2 proteins

(A) Accelerated degradation of the SPRED2^Arg63∗ (R63^∗), SPRED2^Leu100Pro (L100P), and SPRED2^{Leu381Hisfs∗95} (L381Hfs^∗95) proteins. WB analysis shows WT and variant Xpress-tagged SPRED2 protein levels in transfected COS-1 cells, basally and after CHX (10 μg/mL), MG132 (50 μM), or bafilomycin A1 (200 nM) treatment. As shown, accelerated degradation of SPRED2^Arg63∗ mainly occurs via the proteasome, while SPRED2^{Leu381Hisfs∗95} is degraded primarily via autophagy/lysosomes. Both pathways are involved in the accelerated degradation of SPRED2^Leu100Pro, with a more relevant role of the proteasome (slight but statistically significant increased protein level in cells treated with MG132). GAPDH was used as loading control. Representative blots (below) and mean ± SD densitometry values (above) of three independent experiments are shown. Asterisks indicate statistically significant differences (^∗p < 0.05, ^∗∗p < 0.005, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001, ns indicates not significant; two-way ANOVA followed by Sidak’s multiple comparison test).

(B) Subcellular localization of transiently expressed Xpress-tagged WT or mutated SPRED2 proteins in COS-1 cells under steady-state conditions revealed by confocal microscopy analysis. Cells were stained using an anti-Xpress monoclonal antibody and Alexa Fluor 594 goat anti-mouse secondary antibody (red). The F-actin dye Alexa Fluor 488 phalloidin (green) was used to stain the cortical actin associated with the plasma membrane. Merged images with nuclei (DAPI staining, blue) are displayed on the right panels. Scale bar, 10 μm.