Anti ndrg1

Cells were lysed in a RIPA lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 250 mM Na₃VO₄, 10 mM NaF and protease inhibitor cocktail tablet). Western blotting was carried out using the following antibodies: anti-HIF-1α (R&D Systems #MAB1536, mouse monoclonal, 1:1000 dilution), anti-HIF-2α (Thermo Fisher Scientific #PA1-16510, rabbit polyclonal, 1:1000 dilution), anti-HIF-1β (Cell Signaling Technology #5537, rabbit polyclonal, 1:1000 dilution), anti-GLUT1 (glucose transporter 1) (Thermo Fisher Scientific #RB-9052-P, rabbit polyclonal, 1:1000 dilution), anti-Glut3 (AnaSpec #53520, rabbit polyclonal, 1:1000 dilution), anti-β-actin (Sigma #A5441, rabbit polyclonal, 1:5000 dilution), anti-cleaved PARP [poly(ADP-ribose) polymerase] (Cell Signaling Technology #9541, rabbit polyclonal, 1:1000 dilution), anti-PHD2 (Bethyl #A300-322A, rabbit polyclonal, 1:1000 dilution), anti-PHD3 (Bethyl #A300-327A, rabbit polyclonal, 1:1000 dilution), anti-NDRG1 (Cell Signaling Technology #9485, rabbit monoclonal 1:1000 dilution), anti-Cited-2 (Santa Cruz Biotechnology #sc-25375, rabbit polyclonal 1:500 dilution) and hydroxylated P564-HIF-1α (Cell Signaling Technology #3434, rabbit monoclonal 1:1000 dilution). All images are representative of a minimum of three independent experiments, ranging from 3–6.

Equal amounts of protein were resolved by SDS-PAGE and transferred to nitrocellulose membrane using the Invitrogen Nu-Page system (Carlsbad, CA). Western blot analysis was performed using the following protein-/phospho-protein-specific antibodies: anti-p70 S6 kinase (#2708), anti-phospho-p70 S6 kinase (T389) (#9234), anti-rpS6 (#2217), anti-phospho-rpS6(S240/244) (#5364), anti-phospho-NDRG-1 (T346) (#5482), anti-NDRG-1 (#9485), anti-phospho-Akt (S473) (#4060), anti-Akt (#4691), anti-FKBP51 (#12210), anti-FKBP52 (#11826), anti-mTOR (#2972), anti-Rictor (#9476), anti-phospho-Rictor (T1135) (#3806), and anti-Raptor (#2280) were from Cell Signaling Technologies (Danvers, MA). Anti-FKBP25 (ab16654) and anti-FKBP12 (ab2918) were from Abcam. Anti-GAPDH (AM4300) was from Ambion Austin, TX.

Cell lysates were prepared by washing cells with phosphate buffered saline (PBS), then lysed in Igepal lysis buffer (10mM Tris pH 7.5, 0.25M NaCl, 0.5% Igepal) supplemented with Complete™ protease inhibitor cocktail (Roche) at 4°C for 5 min, followed by clarification by centrifugation (3 min, 12,000 rpm). Supernatant was mixed with Laemmli sample buffer and boiled at 100°C, separated by SDS-PAGE and proteins transferred to polyvinylidene difluoride membrane (Immobilon-P, Millipore). Membranes were blocked in 5% milk in PBS/ 0.1% Tween-20, incubated with anti-HIF-1α (BD Transduction Laboratories, clone#610959), anti-NCL (abcam, clone# 22758), anti-NDRG1(Cell signaling, clone#5196) or anti-β-actin (Sigma, clone#A5441) primary antibodies and appropriate HRP-conjugated secondary Mouse antibodies (DAKO, clone#P0447) and Rabbit antibodies (Cytiva, clone#NA934V). Chemiluminescence substrate (West Dura, 34076, Thermo Fisher Scientific) was used to visualize proteins using a ChemiDoc XRS+ imaging system (BioRad). Densitometric analysis was performed using ImageJ software (NIH).

Total protein was harvested in RIPA buffer. Protein extraction and western blotting were performed as described previously^{47 (link)}. Anti-NDRG1 (#9485 1:1000, Cell Signaling Technology, US) and anti-GAPDH (3 µg/ml, MBL Co., Ltd, Japan) primary antibodies were used. Other antibodies are shown in Table S2.