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. 2005 Jul;167(1):107–116. doi: 10.1016/s0002-9440(10)62958-6

Figure 5.

Figure 5

Phosphorylation of TSC1 by GSK3 affects TSC1-TSC2 interaction. A: HEK293T cells were treated with LiCl, LY294002, or control. Lysates were immunoprecipitated by anti-hamartin antibodies, and Western blots were probed with anti-hamartin and anti-phospho-threonine antibodies. B: HEK293T cells were transiently transfected with GSK3 or vector control, and analyzed as in A. C: In vitro kinase assays using in vitro translated TSC1 [wild-type and 2A(T357A/T390A)] and Axin as substrates. Samples were incubated with or without purified GSK3 kinase. Values indicating the absolute amount of Pi incorporation per mole of substrate are shown below. The differences between the GSK3(+) and GSK3(−) samples representing the net Pi incorporation attributable to GSK3 activity are also shown. D: The effects of GSK3 activity on TSC1-TSC2 interaction. HEK293T cells were transfected with the indicated vectors. Twenty-four hours after transfection, cell lysates were immunoprecipitated using anti-TSC1 antibodies, analyzed by SDS-PAGE, and immunoblotted for TSC1 and TSC2 proteins. The bottom panel shows the expression level of TSC2 protein in cell lysates. V, empty vector.