Anti glut1

The cells were collected for immunoblotting analysis as previously described^{38 (link)}. The primary antibodies were as follows: anti-HIF1α (Novus Biologicals, Littleton, CO); anti-HIF2α (Novus Biologicals, Littleton, CO); anti-GLUT1 (Abcam plc, Cambridge, UK); anti-C/EBPβ (Santa Cruz Biotechnology, Santa Cruz, CA); and anti-α-tubulin (Cell Signaling Technology, Danvers, MA). HRP-conjugated anti-rabbit IgG (Bio-Rad Laboratories, Hercules CA) or anti-mouse IgG (Bio-Rad Laboratories) antibodies were used as secondary antibodies. The signals were detected with the ECL Plus reagent (Thermo Fischer Scientific, Waltham, MA) using the chemiluminescence protocol. For detecting the signals of GLUT1 and HIF-2α, SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fischer Scientific) was used.

(S)-tryptophanol-derived oxazoloisoindolinone (SLMP53-1) was synthesized by using the protocol previously described in [24 (link)]. For all experiments, SLMP53-1 was dissolved in dimethyl sulfoxide (DMSO) from Sigma-Aldrich (Sintra, Portugal). Dichloroacetic acid (DCA) from Sigma-Aldrich was dissolved in water. Primary antibodies used in Western blot and/or immunohistochemistry were as follows: anti-α-Tubulin (Sigma-Aldrich, T9026), anti-COX4 (F-8; Santa Cruz Biotechnology, sc-376731), anti-E-Cadherin (G-10; Santa Cruz Biotechnology, sc-8426), anti-GAPDH (6C5; Santa Cruz Biotechnology, sc-32233), anti-GLUT1 (Abcam, ab652), anti-HK2 (3D3; Merck Millipore, MABN702), anti-MCT4 (H-90; Santa Cruz Biotechnology, sc-50329), anti-MMP9 (2C3; Santa Cruz Biotechnology, sc-21733), anti-N-Cadherin (13A9; Santa Cruz Biotechnology, sc-59987), anti-PFKFB3 (ThermoScientific, PA5-21931), anti-SCO2 (ProteinTech, 21223-1-AP), total OXPHOS antibody cocktail (Abcam, ab110413), and anti-VEGF1 (ThermoScientific, MA1-16629). Secondary antibodies used in Western blot were as follows: anti-mouse (Abcam, ab6789) and anti-rabbit (Santa Cruz Biotechnology, sc-2004) horseradish peroxidase-conjugated.

Following antibodies were used at a dilution of 1:1000 except where otherwise stated: Anti-Actin (sc-1616 HRP; 1:10,000), anti-ATP7A (sc-376467; 1:100), anti-normal mouse IgG (sc-2025; 1.4 µg for IP), anti-ERK (sc-514302) from Santa Cruz; anti-GFP (2555), anti-OTULIN (14127; 1.4 µg for IP), anti-IκBα (4814), anti-phospho-IκBα (Ser32/36) (9246), anti-LAMP1 (15665; 1:100), anti-phospho-ERK (Thr202/Tyr204) (9101), anti-normal rabbit IgG (2729; 1.4 µg for IP), anti-EEA1 (3288, 1:100) all CST; anti-SNX27 (ab77799; 1.4 µg for IP and 1:50 for IF), anti-VPS26 (ab23892), anti-VPS35 (ab157220; 1:10,000), anti-MRP4 (ab15602; 1:2500), anti-DGKζ (ab105195; 1:100), anti-GLUT1 (ab15309; 1:100 and ab115730) all Abcam; anti-Beta PIX (07-1450-I; 1:5000), anti-Met1-Ub (MABS199) all Millipore; anti-STEAP3 (17186-1-AP), anti-KIDINS220 (21856-1-AP) all Proteintech; anti-SNX27 (for murine SNX27) gift from W. Hong; anti-Beta-catenin (610153, 1:2000) BD; anti-FLAG M2 (F3165; 1:10000 and 1 µg for IP) Sigma; anti-HOIP (MAB8039; 1:5000) R&D; anti-HA (Core facility monoclonal antibodies HMGU; 25 µl for IP); anti-CD2-APC (17-0029-41; 1:200) eBioscience.

Primary antibodies were γ-H2AX (Abcam, Cambridge, UK, Anti-gamma H2A.X (S139 antibody [9F3], ab26350) or GAPDH (14C10) rabbit IgG mAb (Cell Signaling Technology, Cambridge, UK CST, 2118), anti-ckit (Abcam, ab32363), Anti-eNOS (Cell Signaling Technology, 9572), or PDGFR-b (Abcam, ab69506); anti-beta Actin (Abcam, ab8227), anti-IL6 (Abcam, ab6672), anti-phospho-Rb (Abcam, ab47763), anti-p21 (Abcam, ab109520), or anti-CD163 (Abcam, ab156769); anti-mTOR (Abcam, ab2732), anti-NFkB (Santa Cruz Biotechnologies, Inc., Dallas, TX, USA, sc8008), anti-ATM (Abcam, ab32420), or anti-GLUT1 (Abcam, ab115730); CD34 (Santa Cruz Biotechnologies, Inc., Dallas, TX, USA, sc74499). Secondary antibodies: AF488GAM (Molecular Probes, ThermoFisher Scientific, Waltham, MA, USA; Novus Biogicals, LLC, Centennial, CO, USA, A11029), AF546GAR Molecular Probes, A11035), HRP-anti-mouse IgG (BD Pharmigen^TM (BD Biosciences), San Jose, CA, USA, 7076S), and HRP-anti-rabbit IgG (BD Pharmigen^TM, 7074S).

Cells were washed with ice-cold Phosphate-Buffered Saline (PBS) and then lysed in Triton X-100-containing lysis buffer. The composition of the lysis buffer was as follows: 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, 2.5 mM EDTA, 2.5 mM EGTA, 20 mM NaF, 1 mM Na₃VO₄, 20 mM Sodium β-Glycerophosphate, 10 mM Sodium Pyrophosphate, 0.5% Triton X-100, Roche protease inhibitor cocktail and 0.1% β-Mercaptoethanol. Lysates were precleared by centrifugation before use for Western blotting. Equal amounts of protein were loaded for Western blot analysis. All the following antibodies used were obtained from Cell Signaling Technology: anti-phospho-p38 (Thr180/Tyr182), anti-p38, anti-phospho-SAPK/JNK (Thr183/Tyr185), anti-SAPK/JNK, anti-phospho-ERK1/2 (Thr202/Tyr204), anti-ERK1/2, anti-Mcl-1, anti-phospho-Bad (Ser112), anti-Bad, anti-phospho-MEK1/2, anti-MEK1/2, anti-Grb2, anti-phospho-Akt (Thr308), anti-Akt, anti-phospho-STAT3 (Tyr705), anti-STAT3, anti-calnexin, anti-phospho-p70 S6K1 (Thr389), anti-p70S6K1, except for anti-p27 (BD Biosciences), anti-HIF-1α (BD Transduction Laoratories), anti-GAPDH (US Biological), anti-Glut1 (Abcam), anti-α-tubulin (Molecular Probes), anti-β-actin (Sigma), anti-FLAG M2 (Sigma), anti-V5 (Serotec) and anti-HA (Roche).

Western blotting was performed on cultured cells or tissue samples after the indicated treatments. Cell lysates were collected using a sodium dodecyl sulfate lysis buffer (Beyotime Biotechnology, Shanghai, China). Equal amounts of total protein (approximately 15 μg for cell samples and 60 μg for tissue samples) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Millipore, Billerica, MA, USA). Membranes were blocked using 5% nonfat powdered milk (Sangon, Shanghai, China) in TBS with Tween-20 (TBST) at about 20°C for 1.5 h, incubated with primary antibodies overnight at 4°C, washed three times with TBST, and then incubated with secondary antibodies for 1 h. The signal intensity of protein bands was visualized using an Enhanced Chemiluminescence Detection Kit (Tanon, Shanghai, China). A semi-quantitative evaluation of protein density was performed using ImageJ (Version 1.5.3). The following antibodies were used at a 1:1000 dilution: anti-GGH (Cat. #138495; Abcam, Cambridge, UK), anti-PKM (Cat. #150377; Abcam), anti-GLUT1 (Cat. #115730; Abcam), anti-LDHA (Cat. #52488; Abcam), anti-β-actin (Cat. #4970, Abcam), and anti-GAPDH (Cat. #2118; Cell Signaling, Danvers, MA, USA).