Rictor

Cells were washed three times using cold PBS and lysed in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.1% SDS, 1% NP-40, and 0.5% sodium deoxycholate) containing protease inhibitors. Approximate 30 μg of protein was separated with 10% SDS–PAGE gel and blotted onto nitrocellulose membranes. Then membranes were blocked with 5% skim milk at room temperature for 1 hour and then incubated with primary antibodies against GAPDH (Shanghai Kangchen), RICTOR (Bethyl Laboratories), VEGFA (Abcam), Akt (Cell signaling Technology), mTOR (Cell signaling Technology), HIF1α (Abcam) and HIF2α (Abcam) at 4°C overnight, followed by TBST wash and 1 hour incubation with horseradish peroxidase-conjugated secondary antibodies at room temperature. Protein bands were visualized by a Molecular Imager ChemiDoc XRS System (Bio-Rad Laboratories, Hercules, CA, USA).

Antibodies against the following proteins were used in this study: TMBIM6/BI-1 (1:100, ab51905), and RPL19 (1:1000, ab128648) (all from Abcam, Cambridge, UK); RICTOR (1:2000, A300-459A) (Bethyl Laboratories, Montgomery, TX, USA); AKT (1:1000, #9272), GST (1:1000, #2622), mTOR (1:1000, #2972), mTOR (1:1000, #4517), NDRG1 (1:1000, #9408), phospho-Ser473-AKT (1:1000, #9271), phospho-Ser939-TSC2 (1:1000, #3615), phospho-Thr308-AKT (1:1000, #4056), TSC2 (1:1000, #4308), phospho-Thr346-NDRG1 (1:1000, #3217), SIN1 (1:1000, #12860), p70 S6 Kinase (1:100, #9202), RICTOR (1:1000, #2114), RICTOR (Sepharose bead conjugate, #5379), and RAPTOR (1:1000, #2280) (all from Cell Signaling Technology, Danvers, MA, USA); HA (1:2000, 11867423001) (Roche Diagnostics, Basel, Switzerland); actin (1:1000, sc-47778), Ki-67 (1:100, sc-15402), phospho-Thr450-AKT (1:1000, sc-293094), RPL19 (1:1000, sc-100830), and RPS16 (1:1000, sc-102087), (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA). Secondary antibodies (1:10,000) for immunoblotting (Jackson ImmunoResearch, West Grove, PA, USA) and immunoprecipitation (1:5000, sc-2006) (Santa Cruz Biotechnology) were also used. Insulin (I3769), EGF (E9644), and IGF1 (I3769) were from Sigma-Aldrich. BAPTA-AM (B6769), BAPTA (B1212), and EGTA-AM (E1219) were from Thermo Fisher Scientific (Waltham, MA, USA).

Protein lysates were harvested and the concentrations were determined by PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, USA). Western blot analysis was carried out using a standard procedure, as previously described [14 (link)]. The following primary antibodies were used following the manufacturer’s instructions: RICTOR (Bethyl Laboratories, #A300-459A, TX, USA, 1:2000), AKT (Cell Signaling Technology, #9272S, MA, USA, 1:2000), p-AKT (S473) (Cell Signaling Technology, #4060S, MA, USA, 1:2000), and GAPDH (Santa Cruz, #sc-25778, TX, USA, 1:2000). Antibody detection was carried out using HRP-linked anti-rabbit IgG antibody (Cell Signaling Technology, #7074S MA, USA), followed by the ECL reaction (Millipore, Burlington, MA, USA). The signals from a fluorescent Western blot were quantified using the ImageJ software.

MPMECs were isolated from Rptor^fl/fl mice and transduced with Cre adenovirus for 16-48 hrs. Ad-CMV-Null was used as a control. To assess VEGF-B activation of mTORC1, cells were serum starved in EBM-2 medium supplemented with 0.2% FBS overnight. A time course was performed on cells treated with 300 ng/mL of recombinant mouse VEGF-B₁₈₆ (R&D Systems #767-VE-010) for 0, 5, 15, or 30 minutes prior to harvest. In a separate experiment, cells were incubated with VEGF-B₁₈₆ (0-300 ng/mL) for 30 min before harvest. Pre-cleared lysates were electrophoresed on a SDS-polyacrylamide gel and transferred to nitrocellulose membranes, as previously described^{24 (link)}. The following primary antibodies were used at 1:1000 dilution: Raptor (Cell Signaling Technology (CST) #2280), Rictor (Bethyl #A300-459A), Rictor (Millipore #05-1471), phospho-S6K1 (T389, CST #9234), S6K1 (CST #9202), phospho-S6RP (S235/236, CST #2211), S6RP (CST #2217), phospho-4EBP1 (T37/46, CST #9459), phospho-4EBP1 (T70, CST #9455), 4EBP1 (CST #9644), phospho-AKT (T308, CST #4056), phospho-AKT (S473, CST #4060), AKT (CST #2920), CLSTN1 (Abcam #ab134130), and β-tubulin (Sigma #T4026). Blots were incubated with secondary antibodies IRDye 680LT goat anti-mouse (1:20,000; LI-COR #925-68020) or IRDye 800 CW goat anti-rabbit (1:10,000; LI-COR #925-32211) and imaged using LI-COR Odyssey.

For western blot analysis, protein lysates (10–50 μg) were size-separated by SDS-PAGE (Invitrogen), transferred onto PVDF membranes and probed with the indicated antibodies. Blots were probed with polyclonal rabbit antibodies recognizing p-mTOR (Ser 2448), mTOR, p-p70 S6 kinase (Thr 389), p70 S6 kinase, p-RPS6 (Ser 235/236), RPS6, p-4E-BP1 (Thr 36/45), 4E-BP1, p-AKT (Ser 473), p-AKT (Thr 308), Lyn, eIF4G, Caspase-3 and LC3A/B (Cell Signaling Technology), eIF4E, PARP-1, YWHAZ (Santa Cruz Biotechnology), Raptor, Rictor, MARS (Bethyl Laboratories), AKT (Upstate), polyclonal goat anti-FKBP11 antibody from Santa Cruz Biotechnology, or monoclonal mouse antibodies recognizing β-Actin (Abcam), CDC25A and TLC1A (Santa Cruz Biotechnology). After incubation with appropriate secondary antibodies, signals were detected by enhanced chemiluminescence (Pierce). For co-immunoprecipitation assays, IgG or mTOR antibodies (Cell Signaling Technology) were added to 0.2 mg protein lysates and incubated overnight at 4°C. Following incubation (1 h, 4°C) with Protein A-Sepharose beads (Invitrogen) complexes were washed three times with NT2 buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM MgCl₂, 0.05% NP-40) and analyzed by western blotting.

Human Akt was subcloned into pEBG (J. H. Kehrl, NIAID, NIH) and flag-tagged dominant negative p38α was kindly provided by Dr. J. Han (Xiamen University, China). Lentivirus packaging vector pSPAX2, lentivirus envelop vector pMD2.G, and lentiviral shRNA expressing vector containing shRNA sequences for mTORC1/2 components (pLKO.1-shCTR, shRictor, shSin1, shRaptor) were provided by Dr. T. Gao (University of Kentucky, Lexington KY). Lentivirus was produced by the Department Molecular and Cellular Biochemistry Viral Core (University of Kentucky, Lexington KY).
Commercial antibodies were used: Flag, β-actin (Sigma); phospho-p38, p38, phospho-ERK1/2, ERK1/2, phospho-Akt (Ser473 and Thr308), Akt, phospho-MK2 (Thr334), MK2, phospho-HSP27 (Ser82), Rictor, mTOR, Raptor (Cell Signaling); Rictor (Bethyl); and HSP27, Sin1 (CalBiochem).