Guardian of the genome:

Transcription

1 Guardian of the genome: 53 application guide

2 Introduction The majority of human cancers result from mutations in the tumour suppressor protein, p53, which is encoded by the T53 gene. The p53 protein is overexpressed in a wide variety of transformed cells and is mutated or inactivated in about 50% of cancers. Many important biological functions within the cell are regulated in a p53-dependent manner. cting as a stress sensor, p53 transmits a variety of stress-inducing signals to activate a range of cellular responses and is activated in response to DN damage, hypoxia, metabolic stress and oncogene activation. Following cellular stress, p53 co-ordinates a range of cellular responses such as cell cycle arrest, cellular senescence, autophagy and cell death initiation (see figure 1). DN damage Oncogene activation Metabolic stress Hypoxia Signaling inputs p53 Mdm2 Dram uma/noxa p21 Tigar Transcription targets p53 regulation utophagy Cell death Cell cycle arrest Cell survival Cellular response Figure 1. Cellular stress can result in p53-dependent upregulation of a range of pathways involved in cell death, cell cycle arrest, senescence, and cell survival. 2

3 ctivity In unstressed cells, levels of p53 are kept low due to its continuous targeting for proteasomal degradation by the E3 ubiquitin ligase, Mdm2. However, following cellular stress p53 becomes post-translationally modified resulting in its stabilization. Upon protein stabilization, p53 forms a homodimeric tetramer that binds to transcriptional response elements and can recruit components of the transcriptional machinery to regulate transcription of several hundred RN polymerase II transcribed genes and thousands of others. TM Oncogene p14rf DN damage TR Mdmx KT p53 xin in1 p53 S46 mek ck poptosis Set7/9 Mdm2 p38 Hipk2 roteasomal degradation Bax Bci2 p53 p53 Mitochondrion Mdmx 300/CB CF HDC Cyt C paf-1 Casp 9 TM S333 S15 p53 CK1 ck S20 DK CHK1 DN damage DN-K S37 p53 mer p38 ERK KT Oncogene Cell cycle arrest Cell growth inhibition Cell death MRT E4F1 Metabolic stress adaption Figure 2. ost-translational modification of p53 by a range of kinases, acetylases, methylases and ubiquitylases can determine cell fate. 3

4 ost-translational modification of p53 Targeting of p53 to specific genes is a highly complex process and depends on the multi-post translational status of p is subject to a variety of post-translational modifications which regulate its complex transcriptional activity and its subcellular localization. Following cellular stress, p53 can undergo mono- and poly-ubiquitination, phosphorylation, sumoylation, neddylation, methylation, acetylation and prolyl-isomerisation on multiple sites (see figure 2+3). In healthy cells, p53 is targeted by the E3 ubiquitin-ligases Mdm2, CO1, irh2 and RF- B1, resulting in the export of p53 from the nucleus to the cytosol and its subsequent proteasomal degradation. Mdm2 is thought to inhibit p53 activity in two ways, firstly by ubiquitinating p53, resulting in its proteasomal degradation, and secondly by binding to a region that prevents the interaction of p53 with CB/p300, thus inhibiting its transcriptional activity. Following cellular stress, p53 becomes phosphorylated by a range of serine/threonine kinases including TM, TR, Chk1/2, DN-K, JNK, HIK2 and acetylated by a range of acetylases including p300/cb, CF and TI60 (see figure 2). hosphorylation of the amino terminus of p53 leads to a conformational change that prevents Mdm2 binding, resulting in its stabilization and allowing for increased interaction and acetylation by CB/p300. The histone acetyltransferases CB/p300 and CF acetylate p53 and enhance its transcriptional activity. cetylation of p53 by CB/p300 occurs at multiple lysine residues (K164, 370, 372, 373, 381, 382 and 386) and by CF at K320. Furthermore, acetylation of the C-terminus of p53 is found to increase the activity and stability of p53 by preventing its ubiquitylation and targeting for proteasomal degradation. Residues that can become acetylated are also subject to methylation. Methylation of p53 by Set9 enhances p53-dependent transcription, whereas acetylation of p53 by SMYD2 leads to repression of its transcriptional activity. In response to DN damage, p53 levels are upregulated, allowing p53 to initiate transcription of genes such as XC, p53r2 and DDB2. DN damage can also induce p53-mediated G1 cell cycle arrest through the activation of the Cdk inhibitor, p21. In addition, p53 can also initiate apoptosis through the upregulation of Bax, Fas, paf-1, Bid, Noxa and uma, which eventually leads to mitochondrial outer membrane permeabilization and subsequent cell death. In addition to transcriptional regulation, p53 is also found to translocate to the mitochondria following cell death stimuli, where it interacts with anti-apoptotic proteins, Bcl-2 and Bcl-XL, which results in the release of cytochrome C from the mitochondria and subsequent cell death. 4

5 Ch Chk1 hk1 S9 S9 S S6 S18 S 8 S1 CK2 S15 5 S20 p38 S33 S37 S46 TR TR R DN-K DN N-- K ERK2 TF1 TSS T81 HIK2 HI K2 DYRK2 DY YRK2 2 T149 JNK T150 T155 ur S315 CDK1/2 S366 CF CF F K317 E4F1 E4F F1 K320 0 Mdm2 U K372 K 2 K37 SMYD2 S SM MY M YD2 Set7/9 K370 K37 K 370 T377 S378 K373 K3 K 373 Mdm2 b K381 K381 K K KC KC CB/p300 S376 GSK GSK3β K3β K382 K382 K M Set8 8 K386 K3 K R-Set7 p38 FCT-CK2 KR S392 S3 T38 T T Figure 3. p53 can become acetylated, methylated, phosphorylated and ubiquitinated by a range of enzymes (a = acetylation, m = methylation, p = phosphorylation, ub = ubiquitylation). N K164 K120 K305 p300 CB/p300 TI 60 hmof p53 acetylation, methylation and ubiquitylation sites N DN-K Chk1/2 C hk1 1/2 1/ 2 TM/TR JNK K ERK M K K K2 MKK2 38 p53 phosphorylation sites C C 5 5

6 ntibodies for post-translationally modified p53 ntibody Clone pplications Host Reactivity roduct code nti-p53 [b 240] M Flow Cyt, IHC-Fr, I, ICC/IF, WB, IHC- Ms Hu, Ms, Rat, Dog ab26 nti-p53 [b 1801] M ICC/IF, Flow Cyt, WB, I, ELIS, IHC-, Ms Hu, Rat ab28 RI, IHC-Fr nti-p53 (phospho S6) M WB, IHC-, ICC/IF, I Ms Hu ab32132 [Y179] nti-p53 (phospho S15) ICC/IF, IHC-, I, WB Rb Hu, Ms, Rat ab1431 nti-p53 (phospho S15) WB, IHC-, ICC/IF Rb Hu ab nti-p53 (phospho S15) WB, ELIS, IHC-, I, ICC/IF Rb Hu ab38497 nti-p53 (phospho S20) M WB, ELIS Ms Hu ab [17B6] nti-p53 (phospho S37) WB, ICC/IF Rb Hu, Ms, Rat ab nti-p53 (phospho T81) ELIS, IHC- Rb Hu ab60991 nti-p53 (phospho S315) IHC-, I, ELIS, WB Rb Hu ab30662 nti-p53 (phospho S315) WB, IHC-, ICC/IF Rb Hu ab nti-p53 (phospho S315) IHC-, WB, ICC/IF Rb Hu ab1647 nti-p53 (phospho S315) [18] M WB, ELIS, IHC- Ms Hu ab nti-p53 (phospho S366) WB, ELIS, IHC- Rb Hu ab79291 nti-p53 (phospho S376) WB, ELIS, IHC- Rb Hu ab60021 nti-p53 (phospho S378) WB, ELIS Rb Hu ab79292 nti-p53 (phospho T387) IHC- Rb Hu ab nti-p53 (acetyl K120) M I, WB, ELIS Ms Hu ab78316 [10E5] nti-p53 (acetyl K319) ELIS, ICC/IF Rb Hu, Ms, Rat ab6270 nti-p53 (mono methyl K372) WB, I Rb Hu ab16033 nti-p53 (acetyl K381) ELIS, ICC/IF, IHC- Rb Hu, Ms, Rat ab61241 nti-p53 (acetyl K382) WB, ICC/IF Rb Hu ab37318 nti-p53 (acetyl K386) IHC-, ELIS, ICC/IF Rb Hu, Ms, Rat ab

7 RabMb antibodies for post-translationally modified p53 ntibody Clone pplications Host Reactivity roduct code p53 RabMb M WB,IHC,ICC/IF Rb Hu ab32389 p53 (C-term) RabMb M WB,ICC, FC, I Rb Hu ab32509 p53 (N-term) RabMab M WB, IHC-, ICC, I Rb Hu ab32049 p53 hospho (ps6) RabMb M WB, IHC-, ICC/IF, I Rb Hu ab32132 p53 hospho (ps9) RabMb M WB Rb Hu ab32076 p53 hospho (ps46) RabMb M WB, IHC, ICC, I Rb Hu ab76242 p53 hospho (pt377) RabMb M WB Rb Hu ab p53 hospho (ps392) RabMb M WB, IHC, ICC, I Rb Hu, Ms, Rat ab33889 p53 hospho (ps392) RabMb M WB, I, IHC-, ICC Rb Hu ab p53 cetyl (K305) RabMb M WB Rb Hu, Ms, Rat ab p53 cetyl (K373) RabMb M WB, IHC-, ICC Rb Hu ab62376 p53 cetyl (K382) RabMb M WB,ICC Rb Hu ab

8 Research tools for p53 analysis Sandwich ELIS, In-Cell ELIS and hosphotracer ELIS provide a convenient and efficient way to study post-translational modifications and levels of p53 protein following treatment of cells or tissue extracts. Figure 4. sandwich ELIS measures the amount of antigen between two layers of antibodies (i.e. capture and detection antibody). The antigen to be measured must contain at least two antigenic sites capable of binding to antibody, since at least two antibodies act in the sandwich. Sandwich ELIS MitoSciences p53 assay kits come in standard ELIS format for measuring total or modified p53. ll assays are highly validated in several cell lines by altering total p53 levels and changes in modification state. Levels of total p53, phosphorylated- or acetylated-p53 protein can be assayed using ELIS to accurately quantify p53 protein levels in human cells and tissue lysates. The MitoSciences p53 ELIS range includes sandwich ELIS kits (see figure 4) to measure levels of modified p53 such as; phospho-ser9 (ab131384), phospho-ser46 (ab124532), phospho-ser392 (ab124533), acetyl-k382 (ab133987) and endogenous p53 (ab117995). These ELIS kits use the same capture antibody and allow you to quantify the endogenous levels of p53, phosphorylated and acetylated p53, or p53 levels as a result of treatments to cells or tissues (see Figure 5). 8

9 (a) (b) Figure 5. Determination of total protein, phosphorylated or acetylated p53 levels using ELIS sandwich assays. (a) Relative levels of phosphorylated Ser46 and total protein were determined following treatment of HEK293T or MCF7 cells with either vehicle, etoposide or camptothecin using kit ab and ab (b) MCF7 cells were treated with histone deacetylase inhibitor trichostatin (TC), sirtuin 1 inhibitor Ex527, ND+-dependent deacetylase inhibitor nicotinamide (Nico), or vehicle (Veh) in the presence of camptothecin (Campto, in red) or vehicle (DMSO, in green). cetyl Lysine382 and total p53 protein levels were determined, respectively, using the kits ab and ab Sandwich ELIS kits for p53 analysis Target hosphorylation/cetylation roduct Code p53 ELI S KIT - ab p53 hospho S6 Human ELIS kit Ser6 ab p53 hospho S9 Human ELIS Kit Ser9 ab p53 hospho S46 Human ELIS Kit Ser46 ab p53 hospho S392 Human ELIS Kit Ser392 ab p53 cetyl K373 Human ELIS kit Lys373 ab p53 cetyl K382 Human ELIS kit Lys382 ab

10 In-Cell Human ELIS (ICE) (a) (b) Cell number Vehicle Camptothecin (x10000) IR800 (total p53) IR700 (p53 pser46) Overlay Figure 6 (a) In-Cell Human ELIS (ICE) assays utilize fluorescently labelled IRDye -conjugated antibodies to allow determination of protein levels using the LI-COR Odyssey or erius. (b) Treatment of MCF-7 cells with camptothecin results in an increase in phospho-ser46 and total-p53 levels compared to the vehicle control as analysed by the ICE assay kit, ab

11 MitoSciences ICE technology is used to perform quantitative immunocytochemistry of cultured cells with a near-infrared fluorescent dye-labeled detector antibody. The technique generates quantitative data with specificity similar to Western blotting, but with much greater quantitative precision and higher throughput due to the greater dynamic range and linearity of direct fluorescence detection and the ability to run 96 samples in parallel. Because the p53 antibody is a mouse antibody and the p53 phospho S9, S46 and S392 antibodies are rabbit, the total p53 protein and a phospho-serine can be measured simultaneously in the same well using the cocktail of provided primary antibodies and the cocktail of IRDye -labeled species-specific secondary antibodies when using a LI-COR infrared imager. This method rapidly fixes the cells in situ, stabilizing the in vivo levels of proteins and their post-translational modifications, and thus essentially eliminates changes during sample handling, compared to the preparation of protein extracts. Finally, the p53 phospho Ser9/46/392 and total p53 protein signals can all be normalized to cell amount, measured by the provided Janus Green whole cell stain, to further increase the assay precision. Total or phosphorylated p53 can be measured in stimulated, irradiated or treated cells by probing with either anti-total-p53, anti-phospho-ser 46 (ab128570) or anti-phospho-ser392 - p53 (ab128571) and secondary antibodies conjugated to IRDyes. 53 levels can be then quantitatively measured within cells by using a LI-COR infrared imager (see figure 6 a and b). ICE kits for p53 analysis Target hosphorylation roduct Code p53 total + pser9 Human In-Cell ELIS Kit (IR) Ser9 ab p53 total + pser46 Human In-Cell ELIS Kit (IR) Ser46 ab p53 total + pser392 Human In-Cell ELIS Kit (IR) Ser392 ab

12 hosphotracer ELIS Figure 7. hosphotracer ELIS utilizes 10-acetyl-3,7-dihydroxyphenoxazine to react with horseradish peroxidase in the presence of peroxide to produce a fluorogenic substrate: resorufin, a highly fluorescent molecule. hosphotracer ELIS kits enable you to detect protein phosphorylation with greater sensitivity than Western blotting. In contrast to standard sandwich ELISs, the hosphotracer ELIS kits allow both the sample and assay reagents to be added simultaneously. Easy to use - minimal pipetting, just one wash step Fast - results in 1h 15 min, no need for secondary antibodies Flexible - detection of one or more targets on the same plate Sensitive - more sensitive than Western blots Fluorometric detection - utilizing standard plate readers found in most labs In contrast to other ELIS formats, no antibodies are present on the assay microplate itself, so assays for several different targets can be performed in different wells on the same microplate, allowing for the readout of more than one protein in a single experiment. 12

13 Figure 8. U2OS cells were treated with fresh media containing doxorubicin at various concentrations for 18 hours. Using the hosphotracer kit ab119640, cells were lysed with hosphotracer Lysis Mix and were transferred to replicate wells of a hosphotracer assay plate and analyzed for either phospho-ser 15, or total p53 using the standard hosphotracer protocol. Signal in the wells was determined using a fluorometric plate reader. For instance, endogenous p53 and phospho-ser15 p53 can be analysed on the same plate (see figure 8). Furthermore p53-related signalling pathways can also be investigated including phospho-erk 1/2 (ab119659), phospho-smd3 (ab119670) and phospho-jnk1/2/3 (ab119634), therefore allowing you to investigate numerous signalling pathways simultaneously. hosphotracer ELIS kits for p53 analysis Target hosphorylation roduct Code hosphotracer p53 (ps15) ELIS kit Ser15 ab hosphotracer p53 Total ELIS kit - ab hosphotracer ERK 1/2 Thr202 and Tyr204 ab (pt202/y204)+ total ERK ELIS kit hosphotracer SMD3 (pser423/425) Ser423 and Ser425 ab total GDH ELIS Kit hosphotracer JNK 1/2/3 Thr183 and Tyr185 ab (pt183/y185) ELIS kit 13

14 Featured antibodies nti-erk1 antibody [Y72] (ab32537) RabMb Reactivity Ms, Hu pplications WB, ICC, I, Flow Cyt, IHC-, ICC/IF Swissrot: lternate Names: Extracellular Signal Regulated Kinase 1 antibody, Insulin Stimulated M2 Kinase antibody, p44 MK antibody, Microtubule-associated protein 2 kinase antibody Function: Involved in MK signaling, the initiation and regulation of meiosis, mitosis, and postmitotic functions in differentiated cells by phosphorylating a number of transcription factors such as ELK-1. hosphorylates EIF4EB1; required for initiation of translation. hosphorylates microtubule-associated protein 2 (M2). hosphorylates SZ1 (By similarity). hosphorylates heat shock factor protein 4 (HSF4). nti-tm (phospho S1981) antibody [E1890Y] (ab81292) RabMb Reactivity Human pplications WB, IHC-, ICC/IF, I Swissrot: Q13315 Human lternate Names: -T mutated antibody, taxia telangiectasia gene mutated in human beings antibody, Serine protein kinase TM antibody, TEL1, telomere maintenance 1, homolog antibody Function: Serine/threonine protein kinase which activates checkpoint signaling upon double strand breaks (DSBs), apoptosis and genotoxic stresses such as ionizing ultraviolet light (UV), thereby acting as a DN damage sensor. Recognizes the substrate consensus sequence [ST]-Q. hosphorylates 'Ser-139' of histone variant H2X/H2FX at double strand breaks (DSBs), thereby regulating DN damage response mechanism. I: anti-tm antibody IB: TM (phospho S1981) antibody (ab81292) 14

15 nti-p53 antibody [b240] (b26) Reactivity Hu, Ms, Ra, Do pplications Flow Cyt, IHC-Fr, I, ICC/IF, WB, IHC- Swissrot: lternate Names: ntigen NY-CO-13 antibody, Cellular tumor antigen p53 antibody, Cys 51 Stop antibody Function: cts as a tumor suppressor in many tumor types; induces growth arrest or apoptosis depending on the physiological circumstances and cell type. Involved in cell cycle regulation as a trans-activator that acts to negatively regulate cell division by controlling a set of genes. nti-mdm2 antibody [SM 14] (ab3110) Reactivity Hu, Ms, Rt pplications WB, IHC-Fr, I, ELIS, IHC- Swissrot: Q00987 lternate Names: Double minute 2 protein antibody, E3 ubiquitin-protein ligase Mdm2 antibody, Hdm 2 antibody Function: E3 ubiquitin-protein ligase that mediates ubiquitination of p53/t53, leading to its degradation by the proteasome. Inhibits p53/t53- and p73/t73-mediated cell cycle arrest and apoptosis by binding its transcriptional activation domain. 15

16 Featured antibodies continued... nti-um antibody (ab9643) Reactivity Hu, Ms pplications WB, IHC-, ICC/IF Swissrot: Q9BXH1 lternate Names: BBC 3 antibody, BBC3_HUMN antibody Function: uma is a member of the pro-apoptotic BH3-family of proteins and is upregulated in a p53-dependent manner. uma localises to the mitochondria following cellular stress, where it activates the pro-apoptotic Bcl-2 family members, Bax and Bak, which results in mitochondrial outer membrane permeabilization and subsequent cell death. nti-noxa antibody [114C307] (ab13654) Reactivity Hu, Ms pplications Flow Cyt, WB Swissrot: Flow Cyt, WB lternate Names: dult T cell leukemia derived M responsive antibody, R antibody, R antibody Function: Noxa is a member of the pro-apoptotic BH3-family of proteins. Noxa becomes upregulated in a p53-dependent manner upon cellular stress. Following mitochondrial localisation, Noxa inhibits anti-apoptotic Bcl-2 family members, resulting in subsequent cell death. 16

17 ntibodies ntibody target Clone pplications Host Reactivity roduct code MDM2 ICC/IF, WB Rb Hu ab87134 JNK1+JNK2 WB Rb Hu ab85139 active Caspase 3 Flow Cyt, ICC, ICC/IF, IHC (F fixed), Rb Hu, Ms, Rat, Dm, InMtj, ab13847 IHC-FoFr, IHC-Fr, IHC-, WB RMk, Xl, Zfsh F1 M ICC, ICC/IF, IHC-, WB Rb Hu, Ms ab32372 TM (phospho S1981) ICC/IF, WB Rb Hu ab79891 TM M Flow Cyt, ICC/IF, IHC-, WB, I Ms Hu, Ms, Rat, Mk ab78 TR-ChI grade ICC/IF, I, WB, ChI Rb Hu, Ms ab2905 Bax M Flow Cyt, ICC/IF, I, WB Ms Hu, Ms, Rat ab5714 Bcl2 ICC, IHC-Fr, IHC-, I, WB Rb Hu, Ms, Rat, Cow ab7973 BRC1 M Flow Cyt, ICC/IF, IHC-Fr, I, IHC- Ms Hu ab16780 BRC1 (phospho S1423) WB, ICC/IF Rb Hu ab90528 BRC2 I, WB Rb Hu, Ms ab90541 Casein Kinase 1 delta ELIS, IHC- Goat Hu, Ms ab10877 Cdc14 M ELIS, ICC, I, WB Ms Hu ab10536 Cdc25 M IHC-Fr, IHC-, I, WB Ms Hu, Rat ab2357 Cdk2 ICC/IF, WB Rb Hu, Rat ab64669 Cdk4 ELIS, I, WB Rb Hu, Ms, Rat ab6551 Chk1 WB Goat Hu ab2845 Chk2 (phospho T68) WB Rb Hu ab3501 Chk2 M ICC/IF, I, WB Ms Hu ab3292 Cyclin D1 M ELIS, Flow Cyt, IHC-Fr, IHC-, I, Ms Hu, Ms, Rat ab6152 WB, ICC Cyclin E M Flow Cyt, ICC/IF, IHC-Fr, IHC-, WB Ms Hu ab3927 Cytochrome C M Flow Cyt, IHC-Fr, ICC/IF, WB, IHC- Ms Hu, Ms, Rat, Eq, ig, Dm ab13575 DN Kcs (phospho S2056) ChI, Flow Cyt, ICC/IF, WB Rb Hu ab ChI Grade DN Kcs (phospho T2609) ICC/IF, WB Rb Hu ab4194 DN Kcs M ICC/IF, IHC-, WB Rb Hu, Ms,Rat, rhm ab32566 E2F1-ChI Grade M ChI, IHC-Fr, IHC- Ms Hu, Ms, Rat ab4070 E2F2 WB Chk Hu, Ms ab14292 E2F4 M GS, ICC/IF, IHC-, WB Ms Hu ab17845 E2F5 WB Rb Hu ab22855 E2F6 M ICC/IF, I, WB Ms Hu ab11952 HDC1 ICC/IF, IHC-, WB, I Rb Hu, Ms, Rat ab19845 HDC2 ICC/IF, IHC-Fr, IHC-, WB, I Rb Hu, Ms, Rat ab16032 HDC3 ICC, IF, IHC-, WB, ICC/IF, I Rb Hu, Ms, Rat, InMtj, Zb ab16047 HDC5 IHC-, I, WB, IHC-FoFr Rb Hu, Ms, Rat ab1439 HDC6 WB Rb Hu ab82557 HDC7 WB Rb Hu, Ms ab23657 HIK2 ELIS, ICC/IF, IHC-Fr, IHC-, WB Rb Hu ab28507 JNK1+JNK2 ICC/IF, IHC-Fr, IHC-, WB Rb Hu, Ms ab4821 (phospho T183 + Y185) KT2B / CF - ChI Grade ChI, IHC-, I, WB, ICC/IF Rb Hu, Ms, Dog ab12188 KT3B / p300 - ChI Grade M ChI, ICC/IF, IHC-Fr, IHC-, I, WB, Ms Hu ab14984 IHC-FoFr MDM2 (phospho S186) M WB Ms Hu ab22710 MEK1 (phospho T386) WB Rb Hu, Ms, Rat ab5615 MEK1 ICC/IF, WB Rb Hu, Ms, Rat ab42647 MEK3 I, WB Rb Hu ab2565 MEK7 ICC, ICC/IF, WB Rb Hu, Ms ab18565 MEK7 (phospho S271 + T275) WB Rb Hu ab

18 ntibodies continued... ntibody target Clone pplications Host Reactivity roduct code Noxa M Flow Cyt, WB Ms Hu, Ms ab13654 p21 ICC/IF, IHC-, WB, ELIS Rb Hu, Rat, Cow ab18209 p38 ICC, ICC/IF, IHC-Fr, IHC-FrFl, IHC-, Rb Hu, Ms, Rat ab7952 I, WB CN - roliferation Marker M Flow Cyt, ICC/IF, IHC-Fr, IHC-, I, WB Ms Hu, Ms, Rat, Chk, ab29 Dm, Zfsh IS2 ELIS, IHC-, WB Rb Hu ab4902 IS3 IHC-, WB Rb Hu, Ms ab22856 KC gamma ELIS, ICC, IHC-Fr, WB Rb Rat ab4145 KC gamma (phospho T674) WB Rb Hu, Rat ab5797 uma WB, IHC-, ICC/IF Rb Hu, Ms ab9643 Raf1 (phospho S621) WB Rb Hu ab4767 Raf1 [Y198] M ICC/IF, IHC-, I, WB Rb Hu, Ms, Rat ab32025 Rb (phospho S249) WB Rb Hu ab4788 Rb (phospho T826) WB Rb Hu ab4779 Rb (phospho T821) WB Rb Hu ab4787 Rb - Nuclear Marker M IHC-Fr, I, WB Ms Hu, Ms ab24 SIRT1 ICC/IF, I, WB Rb Hu ab7343 SIRT2 - Nuclear Marker WB Rb Hu ab10659 SIRT3 WB, ICC/IF Rb Hu ab40963 roteins and peptides Species Expression system pplications roduct code p53 protein (Tagged) Human E.Coli FuncS, WˆB ab43615 p53 protein (Tagged) Human Baculovirus infected sf9 cells SDS-GE,WB ab84768 p53 peptide - phospho S315 Human Synthetic peptide Blocking ab28896 p53 peptide - acetyl K382 Human Synthetic peptide Blocking ab

19 Kits pplications roduct code Caspˆase 3 (active) FITC Staining Kit Flow Cyt, Fluorescence Microscopy, FuncS ab65613 Caspase 3 (active) Red Staining Kit Flow Cyt, Fluorescence Microscopy, FuncS ab65617 Caspase 3 ssay Kit (Colorimetric) Enzyme activity ab39401 Caspase Family Colorimetric Substrate Kit lus FuncS ab Caspase 9 (active) FITC Staining Kit Flow Cyt, Fluorescence Microscopy, FuncS ab65615 Caspase 9 (active) Red Staining Kit Flow Cyt, Fluorescence Microscopy, FuncS ab65619 potrack Cytochrome c poptosis ICC ICC/IF ab ntibody Kit cytochrome C Releasing poptosis ssay Kit WB ab65311 anels ntibodies roduct code Breast Cancer Biomarker anel Ki67, Estrogen receptor alpha, ErbB2, p53, ab rogesterone receptor Biochemicals Biological description urity roduct code ifithrin-hbr p53 inhibitor >98% ab

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