Figure 3

Proteosome-mediated degradation of pathogenic TSC1 variants. (A) Immunoblot showing levels of TSC2, wild-type TSC1 and pathogenic TSC1 variants in cells treated with 42 μM MG-132 for 4 hours, or left untreated. The blot was incubated with an antibody against ubiquitin (Ub) to show the effect of the MG-132 treatment. (B) Immunoblot showing levels of TSC2, wild-type TSC1 and the L50P and L117P variants, S6K and T389-phosphorylated S6K (T389) in cells treated with 42 μM MG-132 and 200 nM insulin (+ insulin/MG-132), 42 μM MG-132 only (+ MG-132), 200 nM insulin only (+ insulin), or left untreated (basal). (C) Quantification of the immunoblot signals, showing MG-132-dependent inhibition of proteosome-mediated degradation of the TSC1 L50P and L117P variants. The mean integrated intensities (arbitrary units) of the signals for wild-type TSC1 (black), and the L50P (grey) and L117P (white) variants are shown. MG-132 treatment resulted in a relative increase in the signals for the L50P and L117P variants compared to wild-type TSC1 in both insulin stimulated and unstimulated cells. (D) Quantification of the immunoblot signals showing increased S6K T389 phosphorylation in MG-132 treated cells expressing the L50P or L117P variants. S6K T389 phosphorylation was determined as in Figure 2. (T389/S6K ratio). Compared to MG-132-treated cells expressing wild-type TSC1, MG-132 treatment of cells expressing the L50P and L117P variants increased the T389/S6K ratio.