Anti ubc9

Manufactured by Cell Signaling Technology

Anti-Ubc9 is a primary antibody that targets the protein Ubc9. Ubc9 is the sole E2 conjugating enzyme that facilitates the covalent attachment of the small ubiquitin-like modifier (SUMO) to target proteins. This antibody can be used to detect and study the expression and localization of Ubc9 in various biological samples.

Automatically generated - may contain errors

Lab products found in correlation

7 protocols using anti ubc9

Lysis Conditions for Preserving Ubiquitin Conjugations

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were washed with PBS and then lysed with either the lysis buffer (#9803; Cell Signaling Technology; 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1mM Na₂EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1mM β-glycerophosphate, 1mM Na₃VO₄, 1 μg/mL leupeptin) or the acidic lysis buffer (50 mM HEPES, pH 6.0, 150 mM NaCl, 0.1% (w/v) SDS) supplemented with protease cocktail (Roche). The acidic lysis buffer was used to preserve the ubiquitin or ubiquitin-like conjugations (e.g., UBA3^N8, Ubc12^N8, UbcH7^Ub, Ubc9^SUMO) that are labile in the regular lysis buffer. The supernatant of the lysate was used for Western blotting. The antibodies used in this study include anti-cullin1 (H213; Santa cruz), anti-UBA3 (F-10; Santa cruz), anti-GlyRS (B01P; Abnova or sc-98614; Santa cruz), anti-SerRS (homemade), anti-V5 (R96-CUS; Invitrogen), and anti-Ube2F (pa5-26641; Thermo Fisher). Other antibodies including anti-NEDD8 (#2754), anti-Ubiquitin (#3936), anti-SUMO1 (#4930), anti-Ubc12 (#5641), anti-Ubc9 (#4918), anti-Flag (#2908), anti-UBA1(#4891), anti-UbcH7 (#3848), anti-UBA2 (#8688), anti-APPBP1 (#14321), anti-p27kip (#3698), and anti-α-Tubulin (#3873) are from Cell Signaling.

Mo Z., Zhang Q., Liu Z., Lauer J., Shi Y., Sun L., Griffin P.R, & Yang X.L. (2016). Neddylation requires glycyl-tRNA synthetase to protect activated E2. Nature structural & molecular biology, 23(8), 730-737.

+ Open protocol

+ Expand

Lysis Conditions for Preserving Ubiquitin Conjugations

Check if the same lab product or an alternative is used in the 5 most similar protocols

+ Open protocol

+ Expand

Western Blot Protein Analysis Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

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).

Shangguan X., He J., Ma Z., zhang W., Ji Y., Shen K., Yue Z., Li W., Xin Z., Zheng Q., Cao Y., Pan J., Dong B., Cheng J., Wang Q, & Xue W. (2021). SUMOylation controls the binding of hexokinase 2 to mitochondria and protects against prostate cancer tumorigenesis. Nature Communications, 12, 1812.

+ Open protocol

+ Expand

AMPK and GPR120 Signaling Pathway

Check if the same lab product or an alternative is used in the 5 most similar protocols

Anti-tGFP (TA150039) was purchased from OriGene (Rockville, MD). Anti-AMPKα (#5831), anti-pAMPKα-Thr172 (#2535), Anti-AMPKα1 (#2795), Anti-AMPKα2 (#2757), anti-c-myc (#5605), anti-pc-myc-Ser62 (#13748), anti-UBC9 (#4786), anti-IL-6 (#13797), anti-NaK ATPase (#7074), anti-α-smooth muscle actin (SMA; #56856), anti-SUMO2/3 (#4971), and anti-MCP-1 (#12838) were purchased from Cell Signaling Technology Inc. (Danvers, MA). Anti-β-arrestin 2 (C16D9) rabbit mAb (Cell Signaling Technology) for the western blotting analysis, and sc-365445 (Santa Cruz Biotechnology) for the IP assay. Anti-β-arrestin 1 (D8O3J) rabbit mAb (Cell Signaling Technology). Anti-α-actin (sc-47778) and anti-SUMO1 (sc-5380) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX). Anti-GPR120 (sc-390752) and anti-SUMO2/3 immunoprecipitation (IP) beads (#BK-162) were purchased from Cellskeleton, Inc and antibody (sc-50331) were purchased from Santa Cruz Biotechnology, Inc. GW9508 (G9797) and AICAR (A9978) were purchased from Sigma-Aldrich (St. Louis, MO). DHA (CAS6217-54-5) was purchased from Cayman Chemical Company (Ann Arbor, MI). Anti-CD68 (14-0681-80) was purchased from Affymetrix-ebioscience.

Yan C.H., Liu H.W., Tian X.X., Li J., Ding Y., Li Y., Mei Z., Zou M.H, & Han Y.L. (2022). AMPKα2 controls the anti-atherosclerotic effects of fish oils by modulating the SUMOylation of GPR120. Nature Communications, 13, 7721.

+ Open protocol

+ Expand

Cell and Tissue Lysis and Immunoblotting

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were lysed in RIPA buffer (6 mM Na₂HPO₄, 4 mM NaH₂PO₄, 2 mM EDTA pH 8.0, 150 mM NaCl, 1% NaDOC, 0.1% SDS and 1% NP-40) and tissues were lysed in Tris–SDS buffer (2% SDS, 0.6 M Tris-Cl pH 8.0 and 0.1 M DTT) supplemented with protease inhibitors (Sigma-Aldrich) and 20 mM N-Ethylmaleimide (NEM; Sigma-Aldrich) and sonicated using the Diagenode Bioruptor. Lysates were clarified by centrifugation and protein concentration was measured using Protein Assay Dye reagent concentrate (Bio-Rad, Hercules, CA) according to the manufacturer’s protocol. The following antibodies and concentrations were used: anti-Flag M2; 1:7500 (Sigma-Aldrich), anti-LRH-1; 1:3000 for mouse liver and 1:10,000 for in vitro assay (R&D, Minneapolis, MN), anti-SF-1; 1:1000 (Upstate, EMD Millipore, Billerica, MA), anti-UBC9; 1:1000 (Cell Signaling, Danvers, MA), anti-SUMO1; 1:1000 (Developmental Studies Hybridoma Bank, Iowa City, IA), anti-SUMO2; 1:1000 (Life technologies) and Ubiquitin monoclonal P4D1 antibody; 1:1000 (Cell Signaling), HRP-conjugated anti-βactin 1:2500 (Cell Signaling), and anti-GAPDH 1:5000 (Santa Cruz Biotechnology, Santa Cruz, CA).

Suzawa M., Miranda D.A., Ramos K.A., Ang K.K., Faivre E.J., Wilson C.G., Caboni L., Arkin M.R., Kim Y.S., Fletterick R.J., Diaz A., Schneekloth JS J.r, & Ingraham H.A. (2015). A gene-expression screen identifies a non-toxic sumoylation inhibitor that mimics SUMO-less human LRH-1 in liver. eLife, 4, e09003.

+ Open protocol

+ Expand

Western Blot Analysis of Cellular Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were lysed with RIPA buffer containing a cocktail of protease (Sigma). Protein samples were separated by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) transfer membrane (Hybond-P; GE Healthcare). The nonspecific antibody binding sites were blocked with milk in Tris-buffered saline–Tween 20 and then reacted with the primary antibodies anti-HA (GTX29110 [GeneTex] or 3724 [Cell Signaling]), anti-Ubc9 (4786; Cell Signaling), anti-actin (A5441; Sigma), anti-GFP (GTX26556GeneTex), anti-STAT1 (9175; Cell Signaling), anti-STAT2 (05-693; Upstate), and anti-RIG-I (3743; Cell Signaling). Anti-NS5 and -NS3 antibodies are gifts from Huey-Nan Wu. Blots were treated with horseradish peroxidase-conjugated secondary antibody (Thermo Scientific), and signals were detected by enhanced chemiluminescence (ECL; Millipore).

Su C.I., Tseng C.H., Yu C.Y, & Lai M.M. (2016). SUMO Modification Stabilizes Dengue Virus Nonstructural Protein 5 To Support Virus Replication. Journal of Virology, 90(9), 4308-4319.

+ Open protocol

+ Expand

Characterization of IRTKS and PCBP2 Interactions

Check if the same lab product or an alternative is used in the 5 most similar protocols

Antibodies used were: anti-IRTKS and anti-EEA1 were from Santa Cruz Biotechnology; anti-IRF3, anti-MAVS, anti-K48-polyubiquitin, anti-H3 and anti-Ubc9 were from Cell Signaling Technology; anti-PCBP2 and anti-SUMO2 were from MBL International Corporation; anti-GST, anti-green fluorescent protein (GFP), anti-HA, anti-FLAG, anti-β-actin and anti-His antibodies were from Sigma-Aldrich; Donkey anti-rabbit or anti-mouse secondary antibodies conjugated with Alexa-488, Alexa-594 or Alexa-405 were purchased from Molecular Probes. HRP-conjugated secondary antibodies were from Santa Cruz Biotechnology. Paraformaldehyde, cycloheximide, MG132, ginkgolic acid, isopropyl-β-D-thiogalactopyranoside, 4,6-diamidino-2-phenylindole andpropidiumiodide were from Sigma-Aldrich.

Xia P., Wang S., Xiong Z., Ye B., Huang L.Y., Han Z.G, & Fan Z. (2015). IRTKS negatively regulates antiviral immunity through PCBP2 sumoylation-mediated MAVS degradation. Nature Communications, 6, 8132.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!