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. 2019 Mar 4;218(3):961–976. doi: 10.1083/jcb.201807154

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© 2019 Locke and Thorner

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Figure 5. — Vps21 physically interacts with TORC2. (A) Strain yMLT78 expressing 3xHA-Tor2 from its endogenous locus was transformed with plasmids expressing either FLAG-Ypt7 (pMLT110) or FLAG-Vps21 (pMLT101). The resulting transformants were grown to mid-exponential phase, and expression of the FLAG-tagged proteins was induced with 2% galactose for 4 h. The induced cells were harvested and lysed, and FLAG-tagged proteins were immuno-isolated from the extracts using resin coated with anti-FLAG antibodies as described in Materials and methods. Samples of the bound proteins were resolved by SDS-PAGE on a 4–20% gradient gel analyzed by immunoblotting (IB) with anti-HA and anti-FLAG antibodies. (B) Strain yMLT78 expressing 3xHA-Tor2 from its endogenous locus was transformed with a plasmid expressing FLAG-Vps21 (pMLT101). The resulting transformants were cultured and harvested as in A. Cells were lysed without detergent. Samples were split and treated with buffer containing 0.67% DDM or no detergent to solubilize proteins for 30 min before clarifying lysates by centrifugation, as described in Materials and methods. IP, immunoprecipitation; T, total lysate; P, pellet fraction; S, supernatant fraction. Protein extracts were resolved and analyzed as in A, with the indicated antibodies.