Fig. 3

Purification, identification, and characterization of mTORC2 and its binding partners. a Silver-stained high molecular weight mTORC2 components purified from U87vIII cells stably expressing FLAG-RICTOR are shown. Four large proteins (numbered as 1-4) were analyzed by mass spectrometry after being run on 7 % SDS-PAGE gel, excised, then digested by Trypsin. Mass spectrometry results are summarized in the table on the right. Filamin A (FLNA) and Myosin-9 (MYH9) represent unknown bands 1 and 3, respectively. †Mascot protein score; a protein with score value >21 with >2 unique peptides is considered significant (P <0.05). b Immunoblots of purified proteins from U87vIII cells showing main components of mTORC2 (mTOR, RICTOR, SIN1) including phosphorylated and total FLNA. c Immunoprecipitation of U87vIII cell lysate by protein G Dynabeads coupled with antibodies against main components of mTOR complex 1 and 2. Phosphorylated FLNA was co-immunoprecipitated with anti-mTOR and anti-RICTOR coated beads while these two components of mTORC2 were pulled down with anti-pFLNA coated beads. RAPTOR, an mTORC1-specific component, can be co-immunoprecipitated with anti-mTOR coated beads but not with anti-pFLNA beads