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8353
DUB Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

DUB Antibody Sampler Kit #8353

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DUB Antibody Sampler Kit: Image 1

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 (upper) or CYLD Antibody #4495 (lower).

DUB Antibody Sampler Kit: Image 2

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

DUB Antibody Sampler Kit: Image 3

Western blot analysis of extracts from G-361, JJN-3, and A20 cell lines using A20/TNFAIP3 (D13H3) Rabbit mAb.

DUB Antibody Sampler Kit: Image 4

Flow cytometric analysis of K-562 cells (blue) and Ramos cells (green) using UCHL1 (D3T2E) XP® Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

DUB Antibody Sampler Kit: Image 5

Confocal immunofluorescent analysis of MCF7 cells using HAUSP (D17C6) XP® Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red).

DUB Antibody Sampler Kit: Image 6

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

DUB Antibody Sampler Kit: Image 7

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

DUB Antibody Sampler Kit: Image 8

Immunoprecipitation of CYLD protein from 293 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is CYLD (D1A10) Rabbit mAb. Western blot analysis was performed using CYLD (D1A10) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used a secondary antibody.

DUB Antibody Sampler Kit: Image 9

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

DUB Antibody Sampler Kit: Image 10

Confocal immunofluorescent analysis of HCT 116 cells using USP10 (D7A5) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

DUB Antibody Sampler Kit: Image 11

After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.

DUB Antibody Sampler Kit: Image 12

Western blot analysis of extracts from Jurkat cells, untreated or treated with TPA #4174 (40 nM) and A23187 (2 μM) for various amounts of time, using A20/TNFAIP3 (D13H3) Rabbit mAb.

DUB Antibody Sampler Kit: Image 13

Confocal immunofluorescent analysis of mouse brain using UCHL1 (D3T2E) XP® Rabbit mAb (green). Blue = Hoescht 33342 #4082 (fluorescent DNA dye).

DUB Antibody Sampler Kit: Image 14

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using HAUSP (D17C6) XP® Rabbit mAb.

DUB Antibody Sampler Kit: Image 15

Western blot analysis of extracts from 293T cells, mock-transfected (-) or transfected with a Myc/DDK-tagged cDNA expression construct encoding full-length human UCHL3 (hUCHL3, +), using UCHL3 (D25E6) Rabbit mAb.

DUB Antibody Sampler Kit: Image 16

Immunoprecipitation of USP10 from Hep G2 extracts. Lane 1 is 10% input, lane 2 is USP10 (D7A5) Rabbit mAb, and lane 3 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900. Western blot analysis was perfomed using USP10 (D7A5) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.

DUB Antibody Sampler Kit: Image 17

Western blot analysis of extracts from COS-7 cells, mock transfected (-) or transfected with a Myc-DDK-tagged human A20/TNFAIP3 construct (+), using A20/TNFAIP3 (D13H3) Rabbit mAb.

DUB Antibody Sampler Kit: Image 18

Confocal immunofluorescent analysis of mouse brain using UCHL1 (D8R2I) XP® Rabbit mAb (green). Blue = Hoescht 33342 #4082 (fluorescent DNA dye).

DUB Antibody Sampler Kit: Image 19

Immunohistochemical analysis of paraffin-embedded mouse forestomach using HAUSP (D17C6) XP® Rabbit mAb (left) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (right).

DUB Antibody Sampler Kit: Image 20

Western blot analysis of extracts from 293T cells, either mock transfected (-) or transfected with a Myc/DDK-tagged cDNA expression construct encoding full-length human USP10 (hUSP10-Myc/DDK, +), using USP10 (D7A5) Rabbit mAb.

DUB Antibody Sampler Kit: Image 21

Confocal immunofluorescent analysis of mouse olfactory bulb (left) and pons (right) using UCHL1 (D3T2E) XP® Rabbit mAb #13179 (green) and GFAP (GA5) Mouse mAb (Alexa Fluor® 555 Conjugate) #3656 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).

DUB Antibody Sampler Kit: Image 22

Immunohistochemical analysis of paraffin-embedded mouse brain using HAUSP (D17C6) XP® Rabbit mAb.

DUB Antibody Sampler Kit: Image 23

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

DUB Antibody Sampler Kit: Image 24

Confocal immunofluorescent analysis of DU 145 (positive; left) and LNCaP (negative; right) cells using UCHL1 (D3T2E) XP® Rabbit mAb (green). Blue pseudocolor= DRAQ5® #4084 (fluorescent DNA dye).

DUB Antibody Sampler Kit: Image 25

Immunohistochemical analysis of paraffin-embedded mouse colon using HAUSP (D17C6) XP® Rabbit mAb.

DUB Antibody Sampler Kit: Image 26

Immunohistochemical analysis of paraffin-embedded human non-small cell lung carcinoma using UCHL1 (D3T2E) XP® Rabbit mAb.

DUB Antibody Sampler Kit: Image 27

Immunohistochemical analysis of paraffin-embedded human endometrioid adenocarcinoma using HAUSP (D17C6) XP® Rabbit mAb.

DUB Antibody Sampler Kit: Image 28

Immunohistochemical analysis of paraffin-embedded DU 145 (left; positive) and LNCaP (right; negative) cell pellets using UCHL1 (D3T2E) XP® Rabbit mAb.

DUB Antibody Sampler Kit: Image 29

Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using HAUSP (D17C6) XP® Rabbit mAb

DUB Antibody Sampler Kit: Image 30

Immunohistochemical analysis of paraffin-embedded colorectal adenocarcinoma using UCHL1 (D3T2E) XP® Rabbit mAb in the presence of control peptide (left) or antigen specific peptide (right).

DUB Antibody Sampler Kit: Image 31

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

DUB Antibody Sampler Kit: Image 32

Immunohistochemical analysis of paraffin-embedded human prostate adenocarcinoma using UCHL1 (D3T2E) XP® Rabbit mAb.

DUB Antibody Sampler Kit: Image 33

Western blot analysis of extracts from various cell lines using UCHL1 (D3T2E) XP® Rabbit mAb (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).

DUB Antibody Sampler Kit: Image 34

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with constructs expressing Myc/DDK-tagged full-length human UCHL1 (hUCHL1-Myc/DDK; +), Myc/DDK-tagged full-length human UCHL3 (hUCHL3-Myc/DDK; +), Myc/DDK-tagged full-length human UCHL5 (hUCHL5-Myc/DDK; +), and Myc/DDK-tagged full-length human BAP1 (hBAP1-Myc/DDK; +), using UCHL1 (D3T2E) XP® Rabbit mAb (upper) or DYKDDDDK Tag Antibody #2368 (lower).

DUB Antibody Sampler Kit: Image 35

Western blot analysis of extracts from Ramos and K-562 cells using UCHL1 (D3T2E) XP® Rabbit mAb (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). As expected, UCHL1 protein expression is not detected in K-562 cells.

To Purchase # 8353T
Product # Size Price
8353T
1 Kit  (9 x 20 µl) N/A

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Phospho-CYLD (Ser418) Antibody 4500 20 µl
  • WB
H 108 Rabbit 
STAMBP Antibody 5245 20 µl
  • WB
  • IP
H Mk 50 Rabbit 
A20/TNFAIP3 (D13H3) Rabbit mAb 5630 20 µl
  • WB
  • IP
H Mk M R 82 Rabbit IgG
UCHL1 (D3T2E) XP® Rabbit mAb 13179 20 µl
  • WB
  • IHC
  • IF
  • F
H Mk M R 27 Rabbit IgG
HAUSP (D17C6) XP® Rabbit mAb 4833 20 µl
  • WB
  • IP
  • IHC
  • IF
H Mk M R 135, 140 Rabbit IgG
USP9X Antibody 5751 20 µl
  • WB
Dg H Mk M R 270 Rabbit 
CYLD (D1A10) Rabbit mAb 8462 20 µl
  • WB
  • IP
H Mk M R 108 Rabbit IgG
UCHL3 (D25E6) Rabbit mAb 8141 20 µl
  • WB
H M R 27 Rabbit IgG
USP10 (D7A5) Rabbit mAb 8501 20 µl
  • WB
  • IP
  • IF
H Mk M R 110 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

Product 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 two western blot experiments per primary.

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.

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 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 the carboxy terminus of human UCHL1, 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. (2007) 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, 688-692.
  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.

Pathways & Proteins

Explore pathways + proteins related to this product.

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