Fig. 2: RHEB p.Y35L mutation enhances mTOR activation.

a Control vehicle, wild-type (WT) and p.Y35L mutant RHEB plasmids were transfected into HEK293T cells. Equivalent protein quantities from cell lysates were subjected to immunoblotting to detect phosphorylated S6 (pS6), total S6, RHEB, and α-tubulin. b The protein levels of pS6 were quantified by densitometry and normalized to those of total S6 for comparison, where the pS6/total S6 ratios of the vehicle samples were set as one arbitrary unit. Data are presented as the mean ± s.e.m, n = 4, *P < 0.05 (one-way ANOVA with Tukey’s post test). c One micromolar recombinant WT or p.Y35L mutant RHEB proteins were mixed with 50 nM BODIPY-GTPγS in reaction buffer. The fluorescence intensity was recorded every 10 s at 30 °C (λ_em 485 nm, λ_ex 528 nm). Relative fluorescence was calculated for comparison. Data are presented as the mean ± s.e.m, n = 3, ^*P < 0.05 (two-way repeated-measures ANOVA). d Brain lysates from three non-FCD controls (1: craniocerebral injury, 2: arterial aneurysm, and 3: intracerebral hemorrhage) and FCDII RHEB p.Y35L mutation carriers were subjected to immunoblotting to detect pS6, total S6, RHEB, phosphorylated mTOR (p-mTOR), total mTOR, and α-tubulin. Immunohistochemical analysis of the brain lesion region of the FCDII RHEB p.Y35L mutation carrier (e) and a corresponding brain region from a non-FCD control (f). Scale bars: 100 μm