Fig. 4

mTORC1 activity assay shows dysregulation of mTORC1 activity for the identified variants (A) HEK293T cells (wild type [WT], loss of function [LOF], heterozygous for LOF variant [het fs], or indicated variants) were incubated either for 1 h in amino-acid-free media (?aa) or for 1 h in amino-acid-free media followed by 1 h of normal growth media (+aa). Cells without prior incubation in amino-acid-free media were used as baseline control. Western blots were performed, and the amounts of P-S6K, S6K, P-4EBP1, and 4EBP1 were measured; GAPDH served as a loading control. For 4EBP1, two bands were detected, with the higher band corresponding most likely to the phosphorylated 4EBP1. For both LOF cell lines as well as the p.Tyr393Cys and, to some extent, the p.Asp296Glu cell line, a shift in these two bands can be seen for amino-acid-free media compared to the WT cell line. Quantification of this shift was hindered by the closeness of the bands. (B) Relative quantification of the changes in phosphorylation in S6K upon amino acid deprivation determined by the ratio of P-S6K and S6K in amino-acid-free media or normal growth media from n = 7 biological replicates (????p < 0.0001, ???p < 0.001, ??p < 0.01, and ?p < 0.05, two-way ANOVA followed by Dunnett?s multiple comparison test). (C) Relative quantification of the changes in phosphorylation in 4EBP1 upon amino acid deprivation determined by the ratio of P-4EBP1 and 4EBP1 in amino-acid-free media or normal growth media from n = 7 biological replicates (????p < 0.0001, ???p < 0.001, ??p < 0.01, and ?p < 0.05, two-way ANOVA followed by Dunnett?s multiple comparison test).