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8342
UV Induced DNA Damage Response Antibody Sampler Kit

UV Induced DNA Damage Response Antibody Sampler Kit #8342

Western Blotting Image 1

Western blot analysis of extracts from untreated or UV-treated 293 cells, using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (upper) or Histone H2A.X Antibody #2595 (lower).

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Western Blotting Image 2

Western Blot analysis of HT29 cells, untreated, nocodazole-treated, and lambda phosphotase-treated, using Phospho-cdc25C (Ser216) antibody (upper), and cdc25C (5H9) Rabbit Mab, #4688, (lower).

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Western Blotting Image 3

Western blot analysis of extracts from HeLa, COS, NIH/3T3 and C6 cells, untreated or UV-treated, using Phospho-Chk1 (Ser345) (133D30) Rabbit mAb.

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Western Blotting Image 4

Western blot analysis of extracts from various cell lines, using RPA32 (4E4) Rat mAb.

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Western Blotting Image 5

Western blot analysis of extracts from Jurkat and HEL cells using ATRIP Antibody.

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Western Blotting Image 6

Western blot analysis of Raw264.7, SV-T2 and HT-29 cells that were untreated or UV-treated (50 mJ, 30 min), using Phospho-ATR (Ser428) Antibody. Lambda phosphatase NEB #P0753 (10,000 Units/ml, 1hr) was used to demonstrate the phospho-specificity of the antibody.

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Western Blotting Image 7

Western blot analysis of extracts from various cell types using Microcephalin-1/BRIT1 (D38G5) Rabbit mAb.

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Western Blotting Image 8

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.

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Western Blotting Image 9

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.

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IHC-P (paraffin) Image 10

Immunohistochemical analysis of paraffin-embedded HT-29 cells untreated (left) or UV-treated (right), using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb.

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Western Blotting Image 11

Western blot analysis of GST-cdc25C, unphosphorylated or phosphorylated in vitro by Chk2, using Phospho-cdc25C (Ser216) (63F9) Rabbit mAb (upper) or cdc25C antibody #9522 (lower).

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Flow Cytometry Image 12

Flow cytometric analysis of HeLa cells, untreated (blue) and UV-treated (green), using Phospho-Chk1 (Ser345) (133D3) Rabbit mAb.

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IF-IC Image 13

Confocal immunofluorescent analysis of HeLa cells, untreated (left) or UV-treated (right), using RPA32 (4E4) Rat mAb (green) showing translocation to distinct nuclear foci after UV damage. Actin filaments have been labeled with Alexa Fluor® 555 phalloidin. Blue pseudocolor = DRAQ5™ (fluorescent DNA dye).

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IF-IC Image 14

Confocal immunofluorescent analysis of HeLa cells using ATRIP Antibody (green). Actin filaments have been labeled with Alexa Fluor®555 phalloidin (red).

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Western Blotting Image 15

Western blot analysis of untreated, UV-treated (50 mJ, 30 min) and nocodazole-treated (50 ng/ml, 24hr) Raw264.7 cells, using Phospho-ATR (Ser428) Antibody (upper) and a total ATR antibody (lower).

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IHC-P (paraffin) Image 16

Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb in the presence of control peptide (left) or Phospho-Histone H2A.X (Ser139) Blocking Peptide #1260 (right).

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IHC-P (paraffin) Image 17

Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using Phospho-cdc25C (Ser216) (63F9) Rabbit mAb.

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IF-IC Image 18

Confocal immunofluorescent analysis of C2C12 cells, untreated (left) or UV-treated (right), using Phospho-Chk1 (Ser345) (133D3) Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red).

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IHC-P (paraffin) Image 19

Immunohistochemical analysis of paraffin-embedded human colon carcinoma, using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb.

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IHC-P (paraffin) Image 20

Immunohistochemical analysis of paraffin-embedded human colon carcinoma, showing cytoplasmic localization, using Phospho-cdc25C (Ser216) (63F9) Rabbit mAb.

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IHC-P (paraffin) Image 21

Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb, showing nuclear localization.

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IHC-P (paraffin) Image 22

Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using Phospho-cdc25C (Ser216) (63F9) Rabbit mAb.

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IHC-P (paraffin) Image 23

Immunohistochemical analysis of paraffin-embedded human lung carcinoma untreated (left) or lambda-phosphatase-treated (right), using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb.

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IHC-P (paraffin) Image 24

Immunohistochemical analysis of paraffin-embedded human lung carcinoma, untreated (left) or lambda phosphatase-treated (right), using Phospho-cdc25C (Ser216) (63F9) Rabbit mAb.

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Flow Cytometry Image 25

Flow cytometric analysis of HeLa cells, untreated (blue) or treated with UV (100 mJ, 2hr recovery; green) using Phospho-H2A.X (Ser139) (20E3) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® isotype control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

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IHC-P (paraffin) Image 26

Immunohistochemical analysis of paraffin-embedded human lymph node, using Phospho-cdc25C (Ser216) (63F9) Rabbit mAb in the presence of control peptide (left) or Phospho-cdc25C (Ser216) Blocking Peptide #1190 (right).

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IF-IC Image 27

Confocal immunofluorescent analysis of HeLa cells, untreated (left) or UV-treated (right), using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red).

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb 9718 20 µl
  • WB
  • IHC
  • IF
  • F
H M R Mk 15 Rabbit IgG
Phospho-cdc25C (Ser216) (63F9) Rabbit mAb 4901 20 µl
  • WB
  • IP
  • IHC
H Mk 60 Rabbit IgG
Phospho-Chk1 (Ser345) (133D3) Rabbit mAb 2348 20 µl
  • WB
  • IF
  • F
H M R Mk 56 Rabbit IgG
RPA32/RPA2 (4E4) Rat mAb 2208 20 µl
  • WB
  • IP
  • IF
H M R Hm Mk 32 Rat IgG1
ATRIP Antibody 2737 20 µl
  • WB
  • IP
  • IF
H 82 (isoform 1) Rabbit 
Phospho-ATR (Ser428) Antibody 2853 20 µl
  • WB
H M R Mk 300 Rabbit 
Microcephalin-1/BRIT1 (D38G5) Rabbit mAb 4120 20 µl
  • WB
H M R Mk 100 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 
Anti-rat IgG, HRP-linked Antibody 7077 100 µl
  • WB
Goat 

The UV Induced DNA Damage Response Antibody Sampler Kit offers an economical means of investigating proteins involved in the cellular response to UV-induced DNA damage. The kit contains enough primary and secondary antibody to perform two western blot experiments per primary.

Antibodies detect endogenous levels of the respective target proteins.

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly267 of human ATRIP protein or Ser428 of human ATR protein. Polyclonal antibodies are purified by protein A and peptide affinity chromatography. Monoclonal antibodies are produced by immunizing animals with a recombinant full-length human Maltose Binding Protein-RPA32 fusion protein or a synthetic peptide corresponding to residues surrounding Tyr415 of human Microcephalin-1/BRIT1 protein. Activation state monoclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser139 of human H2A.X protein, Ser216 of human cdc25C protein, or Ser345 of human Chk1 protein, respectively.

Exposure to ultraviolet radiation (UV) has a profound impact on human health and disease (1). Low level UV exposure induces the production of vitamin D and is a key regulator of calcium metabolism. Conversely, overexposure to UV is associated with an increased risk of cancer, immunosuppression, and many eye disorders, such as cataracts. Photons of UV light can directly damage DNA causing thymine dimers and other pyrimidine dimers between adjacent bases (2). Free radicals and reactive oxygen species induced by UV exposure also result in DNA lesions and have been linked to malignant melanoma (3). DNA damage from replicative stress and genotoxic agents like UV activate the ATR-mediated checkpoint pathway and stimulate DNA repair, cell cycle arrest, and apoptosis (4). ATR recruitment to sites of DNA damage and activation depends, at least in part, on interaction with the complex of single-stranded DNA, Replication Protein A (RPA), and direct binding to the ATR-associated adapter protein, ATRIP (5). In addition, the Rad17-RFC and Rad9-Rad1-Hus1 (9-1-1) protein complexes are independently recruited with TopBP1 to fully activate the checkpoint response (6,7). BRIT1 (MCPH1) is required for UV-induced formation of ATR, RPA, and p-Rad17 foci at sites of DNA damage (8-10) and may regulate the expression of several DNA damage response proteins (11). Once activated, ATR phosphorylates a number of mediators, including histone H2AX Ser139 and Chk1 kinase at Ser345. H2AX phosphorylation is a marker of DNA damage. Complete loss of H2AX results in reduced Chk1 activation and impaired survival of cells after UV exposure (12). Chk1 and Chk2 kinase activation is essential for checkpoint-mediated control of cell cycle progression (4). Checkpoint kinases stimulate cell cycle arrest by phosphorylation of a group of tyrosine phosphatases known as Cdc25A, Cdc25B, and Cdc25C (13 -15). Both Chk1 and Chk2 kinases phosphorylate Cdc25C at Ser216 in response to DNA damage and stimulate arrest (16-17).

  1. Peng G et al. (2009) Nat Cell Biol 11, 865–72
  2. von Thaler, A.K. et al. (2010) Exp Dermatol 19, 81-8.
  3. Rastogi, R.P. et al. (2010) J Nucleic Acids 2010, 592980.
  4. Narayanan, D.L. et al. (2010) Int J Dermatol 49, 978-86.
  5. Zou, L. and Elledge, S.J. (2003) Science 300, 1542-8.
  6. Zhou, B.B. and Elledge, S.J. (2000) Nature 408, 433-439.
  7. Blasina, A. et al. (1999) Curr. Biol. 9, 1-10.
  8. Furnari, B. et al. (1999) Mol. Biol. Cell 10, 833-845.
  9. Zou, L. et al. (2002) Genes Dev 16, 198-208.
  10. Mordes, D.A. and Cortez, D. (2008) Cell Cycle 7, 2809-12.
  11. Rai, R. et al. (2006) Cancer Cell 10, 145-57.
  12. Lin, S.Y. et al. (2010) Yonsei Med J 51, 295-301.
  13. Lin, S.Y. et al. (2005) Proc Natl Acad Sci U S A 102, 15105-9.
  14. Revet, I. et al. (2011) Proc Natl Acad Sci U S A 108, 8663-7.
  15. Mailand, N. et al. (2000) Science 288, 1425-9.
  16. Sanchez, Y. et al. (1997) Science 277, 1497-501.
  17. Matsuoka, S. et al. (1998) Science 282, 1893-7.
For Research Use Only. Not For Use In Diagnostic Procedures.

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