Fig. 6.

A: Allelic difference in binding of Tsc2 to Tsc1 and turnover of Tsc2 protein B6 (M552) or BTBR (V552) forms of Tsc2 were introduced into Tsc2^−/− MEFs. TSC complexes were immunoprecipitated and immunoblotted with Tsc1 or Tsc2 antibody. The amounts of Tsc1 and Tsc2 in the complexes were quantified, and the binding efficiency of Tsc1 and Tsc2 is shown in a bar graph. The amount of binding of the BTBR (V552) form of Tsc2 to Tsc1 is set to one (n > 4; P = 0.013). B: Tsc2-expressing cells described above were treated with 100 μg/ml cycloheximide. Cells were harvested 2, 4, and 8 h after treatment. Western blotting was performed to measure the degradation of Tsc2 over time. The quantified Tsc2 signal from three independent experiments is plotted over time (n > 4; P < 0.001). C: Tsc2 protein from the parental mouse livers was detected by Western blotting (four mice from each strain). The levels of Tsc2 were quantified and expressed as a bar graph after correcting for Actin loading control (P = 0.03).