Cell Signaling Technology

Product Pathways - Protein Stability

DUB Antibody Sampler Kit #8353

Kit Includes Quantity Applications Reactivity MW (kDa) Isotype
Phospho-CYLD (Ser418) Antibody #4500 40 µl W H 108 Rabbit
STAMBP Antibody #5245 40 µl W IP H Mk 50 Rabbit
A20/TNFAIP3 (D13H3) Rabbit mAb #5630 40 µl W IP H M R Mk 82 Rabbit IgG
UCHL1 Antibody #3524 40 µl W H M R Mk 27 Rabbit
HAUSP (D17C6) XP® Rabbit mAb #4833 40 µl W IP IF-IC H M R Mk 135, 140 Rabbit IgG
USP9X Antibody #5751 40 µl W H M R Mk Dg 270 Rabbit
CYLD (D1A10) Rabbit mAb #8462 40 µl W IP H M R Mk (Hm) (B) (Dg) (GP) (Hr) 108 Rabbit IgG
UCHL3 (D25E6) Rabbit mAb #8141 40 µl W H M R (B) (Dg) (Hr) 27 Rabbit IgG
USP10 (D7A5) Rabbit mAb #8501 40 µl W IP IF-IC H M R Mk 110 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl Goat

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IF-IC=Immunofluorescence (Immunocytochemistry)
Reactivity Key:  H=Human  M=Mouse  R=Rat  Hm=Hamster  Mk=Monkey  B=Bovine  Dg=Dog  GP=Guinea Pig  Hr=Horse
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Specificity / Sensitivity

CYLD (D1A10) Rabbit mAb recognizes endogenous levels of total CYLD protein. This antibody also detects an unidentified protein of approximately 28 kDa in some cell types. Phospho-CYLD (Ser418) Antibody detects endogenous levels of CYLD protein only when phosphorylated at Ser418. USP9X Antibody recognizes endogenous levels of total USP9X protein and may also cross-react with USP9Y. All other antibodies in this kit detect endogenous levels of total target protein.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using UCHL1 Antibody #3524.

Western Blotting

Western Blotting

Western blot analysis of extracts from 293 cells, untreated (-) or treated (+) with hTNF-α #8902 (20 ng/ml for 20 minutes), using Phospho-CYLD (Ser418) Antibody #4500 (upper) or CYLD Antibody #4495 (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using HAUSP (D17C6) XP® Rabbit mAb #4833.


Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using STAMBP Antibody #5245.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using A20/TNFAIP3 (D13H3) Rabbit mAb #5630.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using USP9X Antibody #5751.


Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using UCHL3 (D25E6) Rabbit mAb #8141.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using CYLD (D1A10) Rabbit mAb #8462.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using USP10 (D7A5) Rabbit mAb #8501.


Description

The DUB Antibody Sampler Kit offers an economical means of evaluating the presence and status of selected DUB enzymes. This kit contains enough primary antibody to perform four western blot experiments per primary.

Source / Purification

Phospho-CYLD (Ser418) Antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser418 of human CYLD protein. The remaining polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly194 of human STAMBP protein, Asp110 of human UCHL1 protein, or Phe2130 of human USP9X protein. Polyclonal antibodies are purified by protein A and peptide affinity chromatography. Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Lys37 of human CYLD protein, the amino terminus of human UCHL3 protein, the amino terminus of human USP10 protein, or to the carboxy terminus of human HAUSP protein or a recombinant protein specific to the amino terminus of human A20/TNFAIP3 protein. A20/TNFAIP3 (D13H3) Rabbit mAb was prepared in collaboration with Dr. Marc Schmidt-Supprian, Max Planck Institute of Biochemistry.

Background

Ubiquitinating enzymes (UBEs) catalyze protein ubiquitination, a reversible process countered by deubiquitinating enzyme (DUB) action (1,2). CYLD deubiquitinase regulates inflammation and cell proliferation by down regulating NF-κB signaling through removal of ubiquitin chains from several NF-κB pathway proteins (3,4). Phosphorylation at Ser418 decreases CYLD deubiquitinase activity and is important for IKKε-driven transformation (5). STAM-binding protein (STAMBP or AMSH) is an endosomal DUB that preferentially displays ubiquitin isopeptidase activity toward K63-linked chains (6,7). The amino-terminus of A20 contains deubiquitinating activity for Lys63 branches, such as those found in TRAF6 and RIP, while the carboxyl-terminus contains ubiquitin ligase activity for Lys48 branches of the same substrates and leads to their degradation (8). Both enzymes have been implicated in neurodegenerative diseases (9-11) and play a role in the regulation of neuronal development and spermatogenesis (10,13,14). UCHL1 binds monoubiquitin and UCHL3 shows affinity for both ubiquitin and NEDD8, a ubiquitin-like molecule (11,12). HAUSP can bind and deubiquitinate the p53 transcription factor and an associated regulator protein Mdm2, thereby stabilizing both proteins (15,16). HAUSP also modifies other ubiquitinated proteins such as members of the FoxO family of forkhead transcription factors and the mitotic stress checkpoint protein CHFR (17,18). USP10 appears to be regulated through both protein-protein interactions and phosphorylation. Interaction of USP10 with Ras-GAP SH3 domain binding protein (G3BP) inhibits its ability to disassemble ubiquitin chains (19). ATM-mediated phosphorylation at Thr42 and Ser337 stabilizes USP10, promoting redistribution from the cytoplasm to the nucleus, where it functions in p53 deubiquitination, stabilization, and activation in response to genotoxic stress (20). USP9X possesses a well-conserved catalytic domain with cysteine peptidase activity, which allows for cleavage of ubiquitin and polyubiquitin conjugates. While USP9X expression has been shown to be critical for normal mammalian development (21-23), many of its substrates are only beginning to be elucidated.

  1. Nijman, S.M. et al. (2005) Cell 123, 773-86.
  2. Nalepa, G. et al. (2006) Nat Rev Drug Discov 5, 596-613.
  3. Regamey, A. et al. (2003) J Exp Med 198, 1959-64.
  4. Glittenberg, M. and Ligoxygakis, P. Fly (Austin) 1, 330-2.
  5. Hutti, J.E. et al. (2009) Mol Cell 34, 461-72.
  6. McCullough, J. et al. (2006) Curr Biol 16, 160-5.
  7. Kim, M.S. et al. (2006) Biochem Biophys Res Commun 351, 612-8.
  8. Lin, S.C. et al. (2008) J Mol Biol 376, 526-40.
  9. Leroy, E. et al. (1998) Nature 395, 451-2.
  10. Kurihara, L.J. et al. (2001) Hum Mol Genet 10, 1963-70.
  11. Osaka, H. et al. (2003) Hum Mol Genet 12, 1945-58.
  12. Wada, H. et al. (1998) Biochem Biophys Res Commun 251, 688-92.
  13. Sakurai, M. et al. (2006) J Cell Sci 119, 162-71.
  14. Kwon, J. (2007) Exp Anim 56, 71-7.
  15. Li, M. et al. (2002) Nature 416, 648-53.
  16. Brooks, C.L. et al. (2007) Oncogene 26, 7262-6.
  17. van der Horst, A. et al. (2006) Nat Cell Biol 8, 1064-73.
  18. Oh, Y.M. et al. (2007) Biochem Biophys Res Commun 357, 615-9.
  19. Soncini, C. et al. (2001) Oncogene 20, 3869-79.
  20. Yuan, J. et al. (2010) Cell 140, 384-96.
  21. Pantaleon, M. et al. (2001) Mech Dev 109, 151-60.
  22. Noma, T. et al. (2002) Mech Dev 119 Suppl 1, S91-5.
  23. Xu, J. et al. (2005) J Neurosci Res 80, 47-55.

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