Mtor antibody

The protein levels of several genes were quantified by western blot. Briefly, total proteins were extracted using RIPA Lysis (Beyotime, Catalog number: P0013C, Beijing, China) according to the manufacturer's protocols. Then, proteins were isolated by 10% SDS-PAGE and electro-transferred on PVDF membranes (Bio-Rad, Hercules, CA, USA). Subsequently, the membranes containing proteins were seriatim incubated with the primary and secondary antibodies for the indicated time, including Phospho-PI3K p85/p55 (Tyr458, Tyr199) Monoclonal Antibody (PI3KY458-1A11, Thermo Fisher), PI3K p85 alpha Monoclonal Antibody (A3-D0) (Catalog #MA5-32917, Thermo Fisher), mTOR Antibody (#2972, Cell Signaling Technology), Phospho-mTOR (Ser2448) (D9C2) XP® Rabbit (mAb #5536, Cell Signaling Technology) and anti-GAPDH (ab8245; Abcam). The second antibodies are goat anti-mouse/rabbit (ab205719 and ab205718; Abcam). The protein signals were visualized using the ECL Western Blotting Substrate (Bio-Rad) and quantified using Image J software (NIH, Bethesda, MA, USA).

The CCK8 kit was purchased from MedChemExpress in the United States, the transwell cell was purchased from Corning in the United States, and the Annexin V-FITC/PI apoptosis kit was purchased from Sigma-Aldrich in the United States. Phospho-Akt (Ser473) antibody, Phospho-Akt (Thr308) antibody, Akt (pan) antibody, mTOR antibody, and Phospho-mTOR (Ser2448) antibody were purchased from Cell Signaling Technology, USA.

All chemicals were obtained from Sigma-Aldrich unless otherwise specified. The following antibodies were used in this study: rabbit anti-UCP1 (Abcam, ab155117, GR3233606-10 1:2000), anti-Parvalbumin (ABclonal, A13538, Lot0054370201 1:1000), anti-Transferrin (Abbkine, ABM40235, 1:1000), anti-pSTST6 (Cell Signaling Tech, 56554, 1:2000), anti-STAT6 (Cell Signaling Tech, 9362, 1:2000), anti-pAKT (Ser473) (Cell Signaling Tech, 4060, 1:2000), anti-pAKT (Thr308) (Cell Signaling Tech, 13038,1:2000), anti-AKT (Cell Signaling Tech, 2920, 1:2000), anti-GAPDH (Santa Cruz, sc32233, 1:1000), anti-GFP (Proteintech, 50430-2-AP, 1:1000), anti-pERK (Cell Signaling Tech, 4370, 1:2000), anti-ERK (Cell Signaling Tech, 4695, 1:2000), anti-pPKC (Abcam, ab180848, 1:2000), anti-PKC (Abcam, ab179522, 1:2000), anti-p4EBP1 (Cell Signaling Tech, 2855, 1:2000), anti-4EBP1 (Cell Signaling Tech, 9452, 1:2000), anti-ACTB (Sigma Aldrich, A3854, 1:10000), anti-Flag (Sigma-Aldrich, F7425, 1:10000), HRP-conjugated goat anti-Rabbit IgG (Cell Signaling Tech, 7074, 1:10000), anti-pGSK-3β (Cell Signaling Tech, 5558, 1:2000), anti-GSK-3β (Cell Signaling Tech, 12456, 1:2000). Rictor antibody (Abcam, ab70374, 1:100), mTOR antibody (Cell Signaling Tech, 2983, 1:100) (Supplementary Table 2).

For in vitro mTOR kinase assay, cells were rinsed once with ice-cold PBS and lysed in ice-cold CHAPS buffer. Cell lysates were incubated at 4 °C for 10 min and the supernatant was collected by centrifuging lysates at 13,000 rpm for 10 min. Two micrograms of mTOR antibody (#2972, Cell Signaling Technology, USA) were added to the 2 mg of cell lysates and incubated with rotation for 2 h at 4 °C. About 20 ml of agarose beads (Pierce, USA) were added and the incubation continued for an additional 1 h. mTOR immunoprecipitates were washed twice with the same lysis buffer and twice with kinase wash buffer (25 mM HEPES at pH 7.4, 20 mM potassium chloride, and 1 mM magnesium chloride). Kinase assays were performed for 15 min at 37 °C in a final volume of 15 ml of mTORC1 kinase buffer (25 mM HEPES at pH 7.4, 50 mM KCl, 10 mM MgCl₂, 500 μM ATP) and 150 ng of S6K1 as a substrate. Reactions were stopped by the addition of 10 ml of sample buffer and boiling for 5 min and analyzed by SDS-PAGE and immunoblotting. In vitro mTORC2 kinase assay was performed by using mTORC2 kinase buffer (25 mM HEPES at pH 7.5, 100 mM potassium acetate, 1 mM MgCl₂, 500 μM ATP) with 100 ng of Akt1 as a substrate.

Western blotting was performed as described in our previous publication [42 (link)]. Rabbit polyclonal mTOR antibody was from Cell Signaling (catalogue #2972, Beverly, MA) and was used at a dilution of 1:1000. Mouse β-actin monoclonal antibody was from Sigma (Beijing, China) and was used at 1:2000 dilution. Intensities of protein bands were measured using NIH ImageJ software [43 (link), 44 (link)].