Anti xpress

Equal amounts of total protein were resolved on SDS–polyacrylamide gels and transferred to a PVDF membrane (0.2 μm pore, Bio-Rad) using a semi-dry electrotransfer (Bio-Rad). Gemin5 was immunodetected using anti-Gemin5 (Novus) antibody or anti-Xpress (Thermo Fisher Scientific). Commercial antibodies were used to detect RPL3 and RACK1 (Santa Cruz). P1 and P2 ribosomal proteins were detected with the monoclonal antibody 3BH5 [63 (link)]. Anti-H3A (abcam) and Histone H2A (Cell Signaling) were a kind gift from Dr. C. Gutierrez and Dr. E. Lecona. Immunodetection of tubulin (Merck) was used as loading control. The appropriate secondary HRP-conjugated antibodies (Thermo Fisher Scientific) were used according to the instructions of the manufacturer.

HEK293T cells were maintained in DMEM high glucose (Thermo Fisher Scientific, USA) at 37°C in a humidified atmosphere with 7% CO₂. Cells were cotransfected with the indicated plasmid combinations using Metafectene (Biontex, Germany) according to the manufacturer’s instructions. Forty-eight hours later, cells were harvested in immunoprecipitation/lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 20 μg/ml leupeptin, 2 μg/ml antipain, 1 μg/ml pepstatin) and incubated on a rotator at 4°C for 30 min. The samples were centrifuged at 14,000 g and 4°C for 20 min. Two milligrams of each lysate was incubated with 40 μl of Anti-FLAG M2 Affinity Gel (Sigma-Aldrich, USA) on a rotator at 4°C for 3 h. Beads were washed three times with 700 μl immunoprecipitation/lysis buffer. Bound proteins were eluted by boiling beads in 2× SDS sample buffer (0.1 M Tris-HCl pH 6.8, 5% 2-mercaptoethanol, 4% SDS, 20% glycerin, bromophenol blue) at 98°C for 10 min. For detection of protein expression, anti-Xpress (Thermo Fisher Scientific), anti-mouse-HRP Trueblot (Rockland), and anti-FLAG HRP (MiliporeSigma) antibodies were used (See also Supplemental Figures).

The following commercially available antibodies were used: anti-HIF-1α (NB100–132, Novus; 10006421, 1:1,000 dilution for IB analysis, Cayman; 1:1,000 dilution for IB analysis, 1:200 for IF analysis; MAB 1536, R&D Systems, 1:1,000 dilution for IB analysis); anti-HIF-2α (NB100–122, Novus, 1:1,000 dilution for IB anlysis); anti-Xpress (R910-25, Invitrogen, 1:5,000 dilution for IB analysis); anti-FLAG (F3165, Sigma, 1:10,000 dilution for IB analysis); anti-methyl-Lys (ab23366, Abcam); anti-CD31 (clone 2H8, MAB1398Z, Millipore, 1:200 dilution for immunohistochemical (IHC) analysis); anti-HA (MMS-101R, Covance, 1:5,000 dilution for IB analysis); anti-VEGF (AF493NA, R&D System, 1:200 dilution for IHC analysis); anti-EPO (sc-7956, 1:1,000 for IB analysis), anti-Brn3b (sc-6026, 1:200 dilution for IHC analysis) from Santa Cruz; anti-LSD1 (#2139, 1:1,000 dilution for IB analysis), anti-hydroxyl-HIF-1α (#3434, 1:5,000 dilution for IB analysis), anti-Caspase3 (#9661, 1:200 dilution for IHC analysis), anti-Ki-67 (#9027, 1:100 dilution for IHC analysis) and anti-SET7/9 antibodies (#2813, 1:1,000 dilution for IB analysis) from Cell Signalling. anti-HIF-1α-K32 methyl antibodies were generated by Abfrontier (South Korea, 1:5,000 dilution for IB analysis).

For immunoprecipitation, cells were lysed in TNET buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1 mM EDTA, and 0.2% Triton X-100) and 1x protease inhibitor cocktail (Roche). Cell lysates were incubated with anti-FLAG M2 affinity resin (Sigma) for 2 h at 4 °C. Resins were collected by centrifugation and washed three times with TNET buffer. Bound proteins were eluted, resolved by SDS-PAGE, and immunoblotted with appropriate antibodies. The following antibodies were used: anti-SIRT1, anti-MYC, anti-GST, anti-GAPDH, anti-HA, and anti-p53 (Santa Cruz Biotechnology); anti-acetyl p53 (Millipore); anti-FLAG and anti-β-actin (Sigma); anti-Xpress (Invitrogen); peroxidase-conjugated AffiniPure goat anti-rabbit and anti-mouse IgGs (Jackson ImmunoResearch); anti-CHFR antiserum was raised against a recombinant His-CHFR. Relative protein levels in the immunoblot figures were quantitated by ImageJ and normalized to either β-actin or GAPDH levels. Values are plotted as the mean ± SEM of at least three independent experiments.