Superose 6 gel filtration analysis of the TSC1-TSC2 complex. (A) Subcellular fractionation of the TSC1-TSC2 complex. HEK 293T cells were disrupted as described in Materials and Methods, to prepare a hypotonic extract (cytosol) and a 100 mM NaCl extract. The distribution of TSC1, TSC2 and T1462-phosphorylated TSC2 (TSC2-T1462) in the 2 extracts was compared to the proteins TOR, FMRP, AKT and 14-3-3ζ by immunoblotting. (B) The cytosolic extract was applied to a Superose 6 gel filtration column and the elution fractions analysed by immunblotting for the presence of TOR, TSC2, TSC1, AKT and 14-3-3ζ. Peak elution fractions of the molecular weight standards are indicated. (C) The 100 mM NaCl extract was separated and analysed as in (B), except that the elution fractions were tested for FMRP instead of AKT.
Hoogeveen-Westerveld et al. BMC Biochemistry 2012 13:18 doi:10.1186/1471-2091-13-18