Extended Data Fig. 6: SHOC2 mutations do not affect protein stability or localization in cells.

a. Flow cytometry analysis of SHOC2-EGFP-chy-mCherry transfected 293T cells to compare SHOC2-EGFP abundance/stability between the various GOF and LOF variants in cells. (i) Flow cytometry SHOC2-EGFP vs mCherry dot plot, overlay of SHOC WT and non-transfected control, mCherry+ gate is indicated. (ii) mCherry+ cells were selected to extract the ratio of EGFP vs mCherry signal per cell, and for both signals the respective median EGFP or mCherry signal from non-transfected cells (grey population) was subtracted as background (BG) (EGFP-BG)/(mCherry-BG) and plotted as histogram normalized to mode to account for various cell numbers between WT and R200E transfected cells. (iii) Bar graph representing rel. SHOC2-EGFP abundance of indicated mutants normalized to SHOC2 WT, data represents mean +/− SD of extracted median (EGFP-BG)/(mCherry-BG) values as described in ii from N = 4 independent transfections (individual values per repeat plotted on bar represent median from 1636 < mCherry+ cells < 40098, Avg 20460 cells). b. Representative immunofluorescence images of SHOC2-EGFP-chy-mCherry transfected 293T cells with WT and corresponding SHOC2 mutants. Overlay images of mCherry and EGFP channels (left) and EGFP channel only images (right) for SHOC2 variants are shown, size bar corresponds to 15 µm for all images. Results are representative of three independent transfections for each condition.