Anti senp2

Cells were lysed in RIPA lysis buffer and the protein concentration of the cell lysates determined by BCA Protein Assay (Thermo Scientific, 23227). Equal amounts of protein were loaded onto SDS-PAGE gel and transferred to PVDF membranes. Western blotting was performed using primary antibodies and secondary antibodies conjugated with HRP. For immunoblotting, the following antibodies were used: anti-SENP2 (Abcam, ab58418, 1:1000), anti-HIF-1α (Cell Signaling Technology, 79233, 1:1000), anti-phospho-threonine (Cell Signaling Technology, 9386, 1:1000), anti-Ubc9 (Cell Signaling Technology, 4786, 1:1000), anti-BrdU (Cell Signaling Technology, 5292, 1:1000), anti-ubiquitin (Cell Signaling Technology, 3936, 1:1000), anti-cleaved-caspase 3 (Cell Signaling Technology, 9661, 1:1000), anti-SUMO1 (Cell Signaling Technology, 4930, 1:1000), anti-SUMO2/3 (Cell Signaling Technology, 4971, 1:1000), anti-SENP1 (Cell Signaling Technology, 11929, 1:1000), anti-SENP3 (Cell Signaling Technology, 5591, 1:1000), anti-HA-Tag (Cell Signaling Technology, 3724, 1:2000), anti-Flag-Tag (Cell Signaling Technology, 14793, 1:2000), anti-hexokinase 1 (Proteintech, 19662-1-AP, 1:1000), anti-hexokinase 2 (Proteintech, 22029-1-AP, 1:1000), anti-VDAC1 (Proteintech, 10866-1-AP, 1:1000), anti-alpha tubulin (Proteintech, 66031-1-Ig, 1:5000), anti-GAPDH (Proteintech, 10494-1-AP, 1:5000).

Bortezomib was purchased from Janssen-Cilag (Buckinghamshire, UK) and was dissolved in dimethyl sulfoxide (DMSO). Anti-hnRNP K antibody was purchased from Cell Signaling Technology (New England Biolabs, Schwalbach, Germany) and Santa Cruz Biotechnology (Santa Cruz, Dallas, TX), anti-pan-SUMO, anti-SUMO-1, anti-SENP1, and anti-SENP2 antibodies were purchased from Abcam (Cambridge, MA), anti-c-Myc and anti-HA antibodies were purchased from Cell Signaling Technology, and anti-GAPDH antibody was purchased from GeneTex (Irvine, CA).

[³H]-labeled estrone sulfate (ES) was purchased from PerkinElmer (Waltham, MA, USA). Membrane-impermeable biotinylation reagent Sulfo-NHS-SS-biotin, streptavidin agarose resin, protein G agarose resin, an anti-mouse HRP-linked antibody, and an anti-rabbit HRP-linked antibody were purchased from Thermofisher Scientific (Waltham, MA, USA). A mouse anti-myc antibody (clone 9E10) was bought from Roche (Indianapolis, IN, USA). Rabbit anti-SUMO2/3, anti-Ubc9, and anti-SENP2 antibodies were purchased from Abcam (Cambridge, MA, USA). Topotecan HCL, ginkgolic acid (GA), and 2-D08 were obtained from Selleck Chemicals (Houston, TX, USA). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).