Anti α tubulin

Cells were washed twice in ice-cold phosphate-buffered saline (PBS) (Sigma St Louis, MO, United States) and lysed in the JS buffer containing: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 2 mg/ml aprotinin, 1 mg/ml pepstatin, 2 mg/ml leupeptin, 10 mM Na₃VO₄. Protein concentration was determined by Bradford assay (Biorad, Hercules, CA, United States). Cell lysates were denatured in Laemmli buffer (2% SDS, 5% β-mercaptoethanol, 0.001% Bromophenol Blue, 10% glycerol) for 5 min at 100°C and then subjected to SDS-PAGE. 12% Acrylamide/bis-acrylamide gels were electroblotted into polyvinylidene difluoride (PVDF) membranes (Millipore Co., Bedford, MA, United States). Membranes were then probed with primary antibodies as indicated. The primary antibodies used were: anti-CA-IX (R&D Systems, Minneapolis, MN. United States); anti-HIF-1α (BD Biosciences, San Jose, United States); anti-α-tubulin (Santa Cruz Biotechnology, CA, United States); anti-β-actin (Santa Cruz Biotechnology, CA, United States). Donkey anti-goat, goat anti-mouse, and goat anti-rabbit (Santa Cruz Biotechnology, CA, United States) were used as secondary antibodies. Band intensity was measured with the Image J program.

Protein samples from heart and brain tissues were isolated following the manufacturer’s instructions (Thermo Scientific, Rockford, IL). Western blots were performed using standard procedures as we described previously [15 (link), 16 (link)]. Anti-PDH E1-alpha subunit (phosphor S293) (1:500) and anti-PDH E1-alpha subunit (1:500) were purchased from Abcam (Cambridge, MA, USA). Anti-PDK2 (1:1000), anti-PDK4 (1:1000) antibodies and anti-α-tubulin (1:2000) antibody are from Santa Cruz Biotechnology (Dallas, TX, USA). The secondary antibodies were from Cell Signaling Technology (Danvers, MA, USA).

λ-Phosphatase treatment,^{19 (link)} GST pull down,^{19 (link)} far western,^{19 (link)} protein extracts preparation,^{12 (link)} immunoprecipitation,^{12 (link)} immunoblotting assays^{12 (link)} and biotinylation assay^{12 (link), 62 (link)} were performed as previously described. Immunoblot analysis was performed using the following antibodies: anti-Flag (Sigma, Cat#F3165), anti-Flag-HRP (Sigma; Cat#A8592), anti-MPM2 (05-368, Millipore), anti-Notch1_Val1744 (Cell Signaling, Danvers, MA, USA, Cat#2421), anti-Notch3 (Cell Signaling; Cat#2889); anti-Notch3 M20 (Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-7424), anti-Pin1 (Santa Cruz Biotechnology; Cat#sc-46660), anti-β-actin (Santa Cruz Biotechnology; Cat#sc-47778), anti-Lck (Santa Cruz Biotechnology; Cat#sc-433), anti-α-tubulin (Santa Cruz Biotechnology; Cat#sc-803), anti-LaminB M20 (Santa Cruz Biotechnology; Cat#sc-6217) and anti-Hes1 (Santa Cruz Biotechnology; Cat#sc-25392). The antibody against the activated Notch3-IC protein (N3_IC-act) was kindly provided by Genentech (South San Francisco, CA, USA). The anti-N3_EC (5E1) antibody was kindly provided by Professor A Joutel.^{62 (link)} The anti-pTα antibody was kindly provided by H von Bohemer.^{63 (link)}

Cell lysates were harvested in NP-40 lysis buffer containing Complete Mini protease inhibitor cocktail (Roche Diagnostics). A small portion of each solid tumor (from NSG mice) or mouse spleen (from HIS mice) was collected in NP-40 lysis buffer containing Complete Mini protease inhibitor cocktail and homogenized using a BioMasher® II Micro Tissue Homogenizer (VWR). Protein quantification was carried out using the Pierce™ BCA Protein Assay Kit (Thermo Fisher) and a FilterMax F5 Multi-Mode Microplate Reader (San Jose, CA, United SA). Equal concentrations of protein were separated in Mini-PROTEAN® TGX™ Precast 4–20% Gels (Bio-Rad, Hercules, CA, USA) and transferred to nitrocellulose membranes. Membranes were blocked in PBS containing 5% milk and 0.1% Tween-20 and incubated with primary antibody overnight at 4°C. The following antibodies were used: anti-Symmetric Di-Methyl Arginine Motif [sdme-RG] (1:1000, Cell Signaling, Danvers, MA, USA), anti-PARP (1:1000, Cell Signaling), anti-β-actin (1:5000, Sigma, St. Louis, MO, USA), and anti-α-tubulin (1:250, Santa Cruz Biotechnology). Secondary antibodies used include IgG HRP anti-rabbit and anti-mouse (1:5000, Promega). Blots were developed using Pierce™ ECL Western chemiluminescence substrate (Thermo Scientific). Images were captured using the Amersham Imager 600 (GE Healthcare Life Sciences).

Western blot (WB) was performed as described previously [42 (link)]. The primary antibodies used in this study were anti-SET7/9 (rabbit monoclonal, C24B1, 1:1000; Cell Signaling Technology, Danvers, MA; and mouse monoclonal, clone 5F2.3, 1:500; Merck Millipore), anti-SREK1IP1 (rabbit polyclonal, 1:200; GeneTex Inc, Irvine, CA), anti-H3K4me1 (No. 39298, rabbit polyclonal, 1:5000; Active Motif, Carlsbad, CA), anti-FLAG (mouse monoclonal M2, 1:10,000; Sigma-Aldrich Japan), and anti-α-Tubulin (mouse monoclonal, 1:400; Santa Cruz Biotechnology, Santa Cruz, CA) antibodies.α-Tubulin was used as an internal control for WB.