Figure 2From: Functional characterisation of the TSC1–TSC2 complex to assess multiple TSC2 variants identified in single families affected by tuberous sclerosis complexResults of the functional assays on the TSC2 variants identified in family 1 (variants I820del and R1772C) and family 2 (variants T993M and L1511H). (A) Interaction between TSC1 and TSC2 variants. TSC1–TSC2 complexes were immunoprecipitated with antibodies specific for exogenous TSC1 from HEK 293 cells over-expressing TSC1 and wild-type TSC2 (wt) or TSC1 and the TSC2 variants. (B) Interaction between TSC1 and TSC2 variants. Ratio of coimmunoprecipitated TSC2:TSC1, as detected by immunoblotting, was estimated by densitometry scanning (Total Scan). (C) In vitro rheb GAP activity of immunoprecipitated TSC1–TSC2 complexes. Rheb-bound GDP/GTP ratios were determined after 90 minutes incubation with the immunoprecipitated wild-type TSC1–TSC2 complex (wt), protein A beads only (control), TSC1 only, or TSC1–TSC2 variant complexes. Rheb-bound GDP/GTP prior to addition of the TSC1–TSC2 complexes is shown (t = 0). (D) Inhibition of S6 phosphorylation in Tsc2 -/- MEFs. Percentage of Tsc2 -/- MEFs transfected with expression constructs encoding TSC1 and wild-type TSC2, or TSC1 and the TSC2 variants, and showing inhibition of S6 phosphorylation. Data from at least 3 separate experiments are shown. (E) TSC2-dependent inhibition of S6K-T389 phosphorylation. S6K, TSC1 and wild-type TSC2 (wt), or S6K, TSC1 and the TSC2 variants, were coexpressed in HEK 293 cells. Phosphorylation of S6K at the T389 position was determined by immunoblotting. A representative example of at least 3 separate experiments is shown. (E) TSC2-dependent inhibition of S6K-T389 phosphorylation. Ratio of total S6K:T389-phosphorylated S6K, as detected by immunoblotting, was estimated by densitometry scanning (Total Scan). Mean ratios relative to TSC2 wild-type (wt) are shown.Back to article page