x
HA-mTOR cDNA, HA-Raptor cDNA (kindly provided by Dr. Sabaini, MIT), FLAG-mTOR cDNA (insertion of PCR product from HA-mTOR cDNA into pCMV vector) and FLAG-D2195A mTOR (generated by site directed mutagensis) were transfected with Lipofectamine (Invitrogen) according to the manufacturer’s instructions. Cells were lysed in buffer containing 40 mM HEPES, 120 mM NaCl, 50 mM NaF, 10 mM β-glycerophosphate, 1 mM EDTA, 10 mM sodium pyrophosphate, 1 mM PMSF, 1 μg/μl aprotinin, 2 μg/μl leupeptin and 0.3% CHAPS. Except for the IP samples, 1% TX-100 was used as a detergent. Then 700–1000 μg of protein was incubated with the respective antibody for 4 h followed by incubation with protein A-conjugated agarose beads.
Antibody immobilized beads (Sigma-Aldrich) were used for FLAG-mTOR. Beads were washed three times with the lysis buffer and resuspended with the sample buffer. Prepared samples were subjected to SDS-PAGE (6–16% gradient) and transferred to NC membranes (Amersham Biosciences). The membranes were blocked with TTBS buffer containing 5% skim milk and incubated overnight at 4 °C with antibodies against ULK1 (Santa Cruz Biotechnology), Rictor (Bethyl Laboratories), actin (MP Biomedicals) and other proteins (Cell Signaling). The membranes were next washed with TTBS, incubated with HRP-conjugated secondary antibodies at room temperature for 1 h, and washed again with TTBS. Antibody-bound membranes were developed using ECL (Thermo Fisher).
Article Produced By
Department of Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea
School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea
School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan 689-798, Republic of Korea
Department of Bioscience and Biotechnology, Sejong University, 98 Kunja-Dong, Kwangjin-Ku, Seoul 143-747, Republic of Korea
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4763238/
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