Nb7160

For immunoblot analysis, cells were rinsed twice with PBS and collected into sample buffer containing 50% Tris-Glycine SDS buffer (Novex), 45% RIPA buffer (20 mM HEPES-KOH pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% Nonidet-P40, and 1% sodium deoxycholate), 5% 2-mercaptomethanol (Nacalai tesque), 1% phosphatase inhibitor (Nacalai tesque), and 1% protease inhibitor (Nacalai tesque). Subsequently, the samples were heated at 95 °C for 5 min, and each sample was loaded onto a 5–20% polyacrylamide gel (Wako). After electrophoresis and the transfer of the gels onto PVDF membranes (Merck Millipore), the membranes were fixed with 4%PFA for 30 min and blocked in Tris-buffered saline containing 0.1% Tween 20 and 5% nonfat dry milk for 30–60 min at room temperature. The membranes were then incubated overnight at 4 °C with primary antibodies for MBP (1:500, Thermo Fisher Scientific, MA1–10837), BCAS1 (1:1000, Bioss antibodies, bs-11462R), α-syn (1:1000, BD Biosciences, 610,787), PDGFRα (1:500, Santa Cruz, sc-338), or anti-β-actin (1:10000, Sigma Aldrich, A5441). This was followed by a 60 min incubation with the appropriate secondary antibodies (Novus, NB7574 and NB7160) at room temperature, with visualization by enhanced chemiluminescence (Nacalai tesque). The density of each band was quantified using Image J software.

Antibodies were: anti-human HIF-1α (clone 54; BD Transduction Laboratories, San Jose, CA; 1:1,000), anti-HIF-2α (AF2886, R&D Systems, Minneapolis, MN; 1:1,000), anti-β-actin (NB600-505, Novus Biologicals, Littleton, CO; 1:5,000), anti-MYC (ab9106, AbCam, Cambridge, MA; 1:5,000) anti-BHLHE41 (a generous gift from Montager and Piccolo28 (link); 1:100), goat anti-mouse HRP (NB7511, Novus Biologicals, Littleton, CO; 1:5,000), goat anti-rabbit HRP (NB7160, Novus Biologicals; 1:5,000) and donkey anti-goat HRP (sc-2020, Santa Cruz Biothenology, Dallas, TX; 1:1,000).
Whole-cell extracts prepared in RIPA buffer with protease inhibitors (cOmplete Protease Inhibitor, Roche) were electrophoresed on 4–12% Bis Tris Plus Bolt gels in MES buffer (Invitrogen). Proteins were transferred to nitrocellulose using an iBlot2 (Invitrogen). Blots were blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 (TBST). Primary and secondary antibodies were diluted in 5% milk in TBST, and all washes were performed with TBST. Blots were rinsed briefly with PBS before the addition of ECL Prime Western Blotting Detection Reagent (Amersham).