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8357
Stress and Apoptosis Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

Stress and Apoptosis Antibody Sampler Kit #8357

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Stress and Apoptosis Antibody Sampler Kit: Image 1

Flow cytometric analysis of HeLa cells, untreated (blue) or UV-treated (green), using Phospho-HSP27 (Ser82) (D1H2F6) XP® Rabbit mAb.

Stress and Apoptosis Antibody Sampler Kit: Image 2

Immunohistochemical analysis of paraffin-embedded 293T cells untreated (left) or UV-treated (right) using Phospho-SAPK/JNK (Thr183/Tyr185) (81E11) Rabbit mAb.

Stress and Apoptosis Antibody Sampler Kit: Image 3

Chromatin immunoprecipitations were performed with cross-linked chromatin from cells starved overnight and treated with Human β-Nerve Growth Factor (hβ-NGF) #5221 (50 ng/ml) for 2h and either Phospho-c-Jun (Ser73) (D47G9) XP® Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR SimpleChIP® using Rat CCRN4L Promoter Primers #7983, rat DCLK1 promoter primers, and SimpleChIP® Rat GAPDH Promoter Primers #7964. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.

Stress and Apoptosis Antibody Sampler Kit: Image 4

Chromatin immunoprecipitations were performed with cross-linked chromatin from PC-12 cells starved overnight and treated with Human β-Nerve Growth Factor (hβ-NGF) #5221 (50 ng/ml) for 2h and either Phospho-c-Jun (Ser73) (D47G9) XP® Rabbit mAb or c-Jun (60A8) Rabbit mAb #9165, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across Dclk1, a known target gene of both Phospho-c-Jun and c-Jun (see additional figures containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product data sheet.

Stress and Apoptosis Antibody Sampler Kit: Image 5

Flow cytometric analysis of HT-29 cells, untreated (blue) or UV-treated (green), using Phospho-p53 (Ser15) (16G8) Mouse mAb compared to a nonspecific negative control antibody (red).

Stress and Apoptosis Antibody Sampler Kit: Image 6

Flow cytometric analysis of Jurkat cells, untreated (blue) or treated with etoposide #2200 (green), using Cleaved Caspase-3(Asp175) (5A1E) Rabbit mAb compared to a nonspecific negative control antibody (red).

Stress and Apoptosis Antibody Sampler Kit: Image 7

Flow cytometric analysis of Jurkat cells, untreated (blue) or etoposide-treated (green), using Cleaved PARP (Asp214) (D64E10) XP® Rabbit mAb.

Stress and Apoptosis Antibody Sampler Kit: Image 8

Flow cytometric analysis of Jurkat cells, untreated (blue) or anisomycin-treated (green), using Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP® Rabbit mAb compared to a nonspecific negative control antibody (red).

Stress and Apoptosis Antibody Sampler Kit: 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.

Stress and Apoptosis Antibody Sampler Kit: Image 10

Confocal immunofluorescent analysis of C2C12 cells, untreated (left) or treated with λ phosphatase (middle), and NIH/3T3 cells (right) using Phospho-HSP27 (Ser82) (D1H2F6) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye). Negative staining in NIH/3T3 cells is in agreement with the observation that NIH/3T3 cells do not express HSP27 under basal conditions (5,7).

Stress and Apoptosis Antibody Sampler Kit: Image 11

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Phospho-SAPK/JNK (Thr183/Tyr185) (81E11) Rabbit mAb in the presence of control peptide (left) or Phospho-SAPK/JNK (Thr183/Tyr185) Blocking Peptide #1215 (right).

Stress and Apoptosis Antibody Sampler Kit: Image 12

Confocal immunofluorescent analysis of HT-29 cells, untreated (left) or UV-treated (right), using Phospho-p53 (Ser15) (16G8) Mouse mAb (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red).

Stress and Apoptosis Antibody Sampler Kit: Image 13

Confocal immunofluorescent images of HT-29 cells, untreated (left) or Staurosporine #9953 treated (right) labeled with Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin #8953 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Stress and Apoptosis Antibody Sampler Kit: Image 14

Confocal immunofluorescent analysis of HeLa cells, untreated (left) or treated with Staurosporine #9953 (right), using Cleaved PARP (Asp214) (D64E10) XP® Rabbit mAb (green). Actin filament were labeled with DY-554 phalloidin. Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Stress and Apoptosis Antibody Sampler Kit: Image 15

Confocal immunofluorescent analysis of COS cells, untreated (left) or anisomycin-treated (right) using Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP® Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red).

Stress and Apoptosis Antibody Sampler Kit: Image 16

Western blot analysis of extracts from untreated or UV+TPA-treated HeLa and COS cells, using Phospho-MAPKAPK-2 (Thr334) (27B7) Rabbit mAb (upper), or MAPKAPK-2 Antibody #3042 (lower).

Stress and Apoptosis Antibody Sampler Kit: Image 17

Immunohistochemical analysis of paraffin-embedded human breast carcinoma, control (left) or λ phosphatase-treated (right), using Phospho-HSP27 (Ser82) (D1H2F6) XP® Rabbit mAb.

Stress and Apoptosis Antibody Sampler Kit: Image 18

Western blot analysis of extracts from 293 cells, untreated or UV-treated, NIH/3T3 cells, untreated or UV-treated and C6 cells, untreated or anisomycin-treated, using Phospho-SAPK/JNK (Thr183/Tyr185) (81E11) Rabbit mAb.

Stress and Apoptosis Antibody Sampler Kit: Image 19

Flow cytometric analysis of HeLa cells, untreated (blue) or UV treated (green), using Phospho-c-Jun (Ser73) (D47G9) XP® Rabbit mAb.

Stress and Apoptosis Antibody Sampler Kit: Image 20

Western blot analysis of extracts from HT29 cells, untreated or UV-treated (100 mJ/cm2, 1 hr), using Phospho-p53 (Ser15) (16G8) Mouse mAb (upper) or p53 (DO-7) Mouse mAb #48818 (lower).

Stress and Apoptosis Antibody Sampler Kit: Image 21

Immunohistochemical analysis of paraffin-embedded mouse embryo, using Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb in the presence of control peptide (left) or Cleaved Caspase-3 (Asp175) Blocking Peptide (#1050) (right).

Stress and Apoptosis Antibody Sampler Kit: Image 22

Immunohistochemical analysis of paraffin-embedded human tonsil using Cleaved PARP (Asp214) (D64E10) XP® Rabbit mAb.

Stress and Apoptosis Antibody Sampler Kit: Image 23

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP® Rabbit mAb.

Stress and Apoptosis Antibody Sampler Kit: Image 24

Immunohistochemical analysis of paraffin-embedded mouse lung using Phospho-HSP27 (Ser82) (D1H2F6) XP® Rabbit mAb.

Stress and Apoptosis Antibody Sampler Kit: Image 25

Confocal immunofluorescent analysis of mouse small intestine, untreated (left) or treated with λ-phosphatase (right), using Phospho-c-Jun (Ser73) (D47G9) XP® Rabbit mAb #3270 (green). Actin filaments have been labeled with DY-554 phalloidin (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).

Stress and Apoptosis Antibody Sampler Kit: Image 26

Immunohistochemical analysis using Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb on SignalSlide® Cleaved Caspase-3 IHC Controls #8104 (paraffin-embedded Jurkat cells, untreated (left) or etoposide-treated (right)).

Stress and Apoptosis Antibody Sampler Kit: Image 27

Western blot analysis of extracts from HeLa cells, untreated or treated with Staurosporine #9953 (1 μM, 3 hr), Jurkat cells, untreated or etoposide-treated (25 μM, overnight), and THP-1 cells, untreated or cycloheximide-treated (CHX, 10 μg/ml, overnight) followed by treatment with TNF-α #8902 (20 ng/ml, 4 hr), using Cleaved PARP (Asp214) (D64E10) XP® Rabbit mAb (upper), or total PARP Antibody #9542 (lower).

Stress and Apoptosis Antibody Sampler Kit: Image 28

Immunohistochemical analysis of paraffin-embedded mouse colon using Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP® Rabbit mAb.

Stress and Apoptosis Antibody Sampler Kit: Image 29

Immunohistochemical analysis of paraffin-embedded HeLa cell pellets, control (left) or UV-treated (right), using Phospho-HSP27 (Ser82) (D1H2F6) XP® Rabbit mAb.

Stress and Apoptosis Antibody Sampler Kit: Image 30

Confocal immunofluorescent analysis of HeLa cells, untreated (left) or anisomycin-treated (right), using Phospho-c-Jun (Ser73) (D47G9) XP® Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red).

Stress and Apoptosis Antibody Sampler Kit: Image 31

Immunohistochemical staining of paraffin-embedded mouse embryo, showing cytoplasmic localization in apoptotic cells, using Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb.

Stress and Apoptosis Antibody Sampler Kit: Image 32

Immunohistochemical analysis of paraffin-embedded 293T cell pellets, untreated (left) or UV-treated (right), using Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP® Rabbit mAb.

Stress and Apoptosis Antibody Sampler Kit: Image 33

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Phospho-HSP27 (Ser82) (D1H2F6) XP® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).

Stress and Apoptosis Antibody Sampler Kit: Image 34

Immunohistochemical analysis of paraffin-embedded human breast carcinoma, control (left) or lambda phosphatase-treated (right), using Phospho-c-Jun (Ser73) (D47G9) XP® Rabbit mAb.

Stress and Apoptosis Antibody Sampler Kit: Image 35

Immunoprecipitation of extracts from Jurkat cells, untreated or etoposide-treated (25uM, 5hrs), using Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb. Western blot was performed using the same antibody.

Stress and Apoptosis Antibody Sampler Kit: Image 36

Western blot analysis of extracts from COS and 293 cells, untreated or UV-treated, using Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP® Rabbit mAb (upper) or p38 MAPK Antibody #9212 (lower).

Stress and Apoptosis Antibody Sampler Kit: Image 37

Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using Phospho-HSP27 (Ser82) (D1H2F6) XP® Rabbit mAb.

Stress and Apoptosis Antibody Sampler Kit: Image 38

Immunohistochemical analysis of parafin-embedded human colon carcinoma using Phospho-c-Jun (Ser73) (D47G9) XP® Rabbit mAb in the presence of control peptide (left) or Phospho-c-Jun (Ser73) Blocking Peptide (right).

Stress and Apoptosis Antibody Sampler Kit: Image 39

Western blot analysis of extracts from C6 (rat), NIH/3T3 (mouse), and Jurkat (human) cells, untreated or treated with staurosporine #9953 (1uM, 3hrs) or etoposide #2200 (25uM, 5hrs) as indicated, using Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb.

Stress and Apoptosis Antibody Sampler Kit: Image 40

Western blot analysis of extracts from HeLa or HT-29 cells, untreated (-) or treated (+) with either UV (40 mJ/cm2 with 30 min recovery) or anisomycin (25 μg/mL, 30 min), using Phospho-HSP27 (Ser82) (D1H2F6) XP® Rabbit mAb.

Stress and Apoptosis Antibody Sampler Kit: Image 41

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Phospho-c-Jun (Ser73) (D47G9) XP® Rabbit mAb.

Stress and Apoptosis Antibody Sampler Kit: Image 42

Western blot analysis of extracts from NIH/3T3 or C6 cells, untreated or UV-treated, using Phospho-c-Jun (Ser73) (D47G9) XP® Rabbit mAb (upper) or c-Jun (60A8) Rabbit mAb #9165 (lower).

To Purchase # 8357T
Product # Size Price
8357T
1 Kit  (8 x 20 µl) $ 554

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Phospho-MAPKAPK-2 (Thr334) (27B7) Rabbit mAb 3007 20 µl
  • WB
H M R Mk 49 Rabbit IgG
Phospho-HSP27 (Ser82) (D1H2F6) XP® Rabbit mAb 9709 20 µl
  • WB
  • IHC
  • IF
  • F
H M 27 Rabbit IgG
Phospho-SAPK/JNK (Thr183/Tyr185) (81E11) Rabbit mAb 4668 20 µl
  • WB
  • IP
  • IHC
H M R Dm Sc 46, 54 Rabbit IgG
Phospho-c-Jun (Ser73) (D47G9) XP® Rabbit mAb 3270 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
  • ChIP
H M R Mk Pg 48 Rabbit IgG
Phospho-p53 (Ser15) (16G8) Mouse mAb 9286 20 µl
  • WB
  • IF
  • F
H 53 Mouse IgG1
Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb 9664 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
H M R Mk 17, 19 Rabbit IgG
Cleaved PARP (Asp214) (D64E10) XP® Rabbit mAb 5625 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
H Mk 89 Rabbit IgG
Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP® Rabbit mAb 4511 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
H M R Mk Mi Pg Sc 43 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 
Anti-mouse IgG, HRP-linked Antibody 7076 100 µl
  • WB
Horse 

Product Description

The Stress and Apoptosis Antibody Sampler Kit provides an economical means of evaluating stress and apoptotic responses of each protein. The kit contains enough primary and secondary antibody to perform two western blot experiments per primary antibody.

Specificity / Sensitivity

Each antibody in the Stress and Apoptosis Antibody Sampler Kit detects endogenous levels of target protein. Antibodies do not cross-react with any isoforms or phosphorylation sites of the target protein.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide correspond- ing to amino-terminal residues adjacent to Asp175 of human Caspase-3 or residues surrounding Asp214 of human PARP. Phospho-specific monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser82 of human HSP27, Ser73 of human c-Jun, Thr334 of human MAP- KAPK-2, Ser15 of human p53, Thr180/Tyr182 of human p38 MAPK, or Thr183/Tyr185 of human SAPK/JNK.

Background

Cells respond to environmental or intracellular stresses through various mechanisms ranging from initiation of prosurvival strategies to activation of cell death pathways that remove damaged cells from the organism. Many of the proteins and cellular processes involved in normal signaling and survival pathways also play dual roles in cell death-promoting mechanisms. Apoptosis is a regulated cellular suicide mechanism characterized by nuclear condensation, cell shrinkage, membrane blebbing, and DNA fragmentation. Caspase-3 (CPP-32, Apoptain, Yama, SCA-1) is a critical executioner of apoptosis, as it is either partially or totally responsible for the proteolytic cleavage of many key proteins such as the nuclear enzyme poly (ADP-ribose) polymerase (PARP) (1). PARP appears to be involved in DNA repair in response to environmental stress (2). This protein can be cleaved by many ICE-like caspases in vitro (3,4) and is one of the main cleavage targets of caspase-3 in vivo (5,6). PARP helps cells to maintain their viability; cleavage of PARP facilitates cellular disassembly and serves as a marker of cells undergoing apoptosis (7). The p53 tumor suppressor protein plays a major role in cellular response to DNA damage and other genomic aberrations. Activation of p53 can lead to either cell cycle arrest and DNA repair or apoptosis (8). DNA damage induces phosphorylation of p53 at Ser15 and Ser20 and leads to a reduced interaction between p53 and its negative regulator, the oncoprotein MDM2 (9). MDM2 inhibits p53 accumulation by targeting it for ubiquitination and proteasomal degradation (10,11). Stress-activated protein kinases (SAPK)/Jun amino-terminal kinases (JNK) are members of the MAPK family that are activated by a variety of environmental stresses, inflammatory cytokines, growth factors, and GPCR agonists. Stress signals are delivered to this cascade by small GTPases of the Rho family (Rac, Rho, cdc42) (12). SAPK/JNK, when active as a dimer, can translocate to the nucleus and regulate transcription through its effects on c-Jun, ATF-2, and other transcription factors (12,13). c-Jun is a member of the Jun Family, containing c-Jun, JunB, and JunD, and is a component of the transcription factor AP-1 (activator protein-1). Extracellular signals from growth factors, chemokines, and stress activate AP-1-dependent transcription. The transcriptional activity of c-Jun is regulated by phosphorylation at Ser63 and Ser73 through SAPK/JNK (reviewed in 14). AP-1 regulated genes exert diverse biological functions including cell proliferation, differentiation, and apoptosis, as well as transformation, invasion and metastasis, depending on cell type and context (13, 15-17). p38 MAP kinase (MAPK), also called RK (18) or CSBP (19), is the mammalian orthologue of the yeast HOG kinase that participates in a signaling cascade controlling cellular responses to cytokines and stress (17-20). MKK3, MKK6, and SEK activate p38 MAP kinase by phosphorylation at Thr180 and Tyr182. MAPKAPK-2 is a direct target of p38 MAPK (17). Multiple residues of MAPKAPK-2 are phosphorylated in vivo in response to stress. However, only four residues (Thr25, Thr222, Ser272 and Thr334) are phosphorylated by p38 MAPK in an in vitro kinase assay (21). Phosphorylation at Thr222, Ser272, and Thr334 appears to be essential for the activity of MAPKAPK-2 (6). Heat shock protein (HSP) 27 is one of the small HSPs that are constitutively expressed at different levels in various cell types and tissues. In response to stress, the expression level of HSP27 increases several-fold to confer cellular resistance to the adverse environmental change. HSP27 is phosphorylated at Ser15, Ser78, and Ser82 by MAPKAPK-2 as a result of the activation of the p38 MAP kinase pathway (19,22).

  1. Fernandes-Alnemri, T. et al. (1994) J Biol Chem 269, 30761-4.
  2. Satoh, M.S. and Lindahl, T. (1992) Nature 356, 356-8.
  3. Lazebnik, Y.A. et al. (1994) Nature 371, 346-7.
  4. Cohen, G.M. (1997) Biochem J 326 ( Pt 1), 1-16.
  5. Nicholson, D.W. et al. (1995) Nature 376, 37-43.
  6. Tewari, M. et al. (1995) Cell 81, 801-9.
  7. Oliver, F.J. et al. (1998) J Biol Chem 273, 33533-9.
  8. Levine, A.J. (1997) Cell 88, 323-31.
  9. Shieh, S.Y. et al. (1997) Cell 91, 325-34.
  10. Chehab, N.H. et al. (1999) Proc Natl Acad Sci U S A 96, 13777-82.
  11. Honda, R. et al. (1997) FEBS Lett 420, 25-7.
  12. Kyriakis, J.M. and Avruch, J. (2001) Physiol Rev 81, 807-69.
  13. Leppä, S. and Bohmann, D. (1999) Oncogene 18, 6158-62.
  14. Davis, R.J. (2000) Cell 103, 239-52.
  15. Shaulian, E. and Karin, M. (2002) Nat Cell Biol 4, E131-6.
  16. Weiss, C. and Bohmann, D. (2004) Cell Cycle 3, 111-3.
  17. Rouse, J. et al. (1994) Cell 78, 1027-1037.
  18. Han, J. et al. (1994) Science 265, 808-11.
  19. Lee, J.C. et al. (1994) Nature 372, 739-46.
  20. Freshney, N.W. et al. (1994) Cell 78, 1039-49.
  21. Ben-Levy, R. et al. (1995) EMBO J. 14, 5920-5930.
  22. Landry, J. et al. (1992) J. Biol. Chem. 267, 794-803.

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