FIGURE 1.

Mass spectrometric identification of RNA-binding proteins associated with S6K2. A, multiple nuclear proteins associate with EE-p54S6K2 but not EE-p70S6K1. Nuclear fractions of EE-p70S6K1 and EE-p54S6K2 Hek293 cells, respectively, were isolated and subjected to affinity purification using antibodies against EE tag. Isolated protein complexes were analyzed by SDS-PAGE followed by silver staining. Control precipitations were carried out in the absence of antibodies. B, addition of EE peptide to EE-p70S6K2 nuclear precipitates elutes bound S6K2 and S6K2 interacting proteins. Three elutions (10 mg of EE peptide in 30 μl of lysis buffer) were made in sequence (lanes 1–3), with lane 1 being the first elution, followed by lane 2 and lane 3. The beads left after elution were also analyzed as a control. C and D, RNA-associated proteins were identified by NanoLC-MS/MS to interact with nuclear EE-p54S6K2. Matches with a significance p threshold of <0.05 are shown in D). IP, immunoprecipitation; Con., control.