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. 2010 Mar 22;285(22):17065–17076. doi: 10.1074/jbc.M109.078782

FIGURE 2.

FIGURE 2.

S6K2 and the mTOR-Raptor complex associate with hnRNPs U, F/H, and C1/C2. A, hnRNPs U, F/H, and C1/C2 co-precipitate with nuclear EE-p54S6K2. Exponentially growing EE-p54S6K2 cells were harvested followed by nuclear fractionation. Nuclear immunoprecipitations (IP) were achieved with antibodies against the EE tag of overexpressed S6K2. Control purification was achieved by incubating equal amount of nuclear fraction with protein A-Sepharose beads bound to an unrelated antibody. B, antibodies against hnRNPs U, F/H, and C1/C2 were used, respectively, to co-precipitate EE-p54S6K2 using nuclear fractions as in A. C, hnRNPs co-precipitate endogenous S6K2. Hek293 nuclear lysates was used along with antibodies specific to individual hnRNPs. Western blots (WB) of co-precipitated proteins revealed that S6K2, but not S6K1, co-precipitates with hnRNPs. D, mTOR and Raptor associate with hnRNPs. Antibodies specific to hnRNPs were used to purify immunocomplexes in Hek293 nuclear fractions. Antibodies against mTOR and Raptor were used in a Western blot to determine association.