Rabbit polyclonal anti cleaved caspase 3

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom

Rabbit polyclonal anti-cleaved caspase-3 is a laboratory reagent used to detect the presence of the cleaved form of caspase-3 protein, a key executioner of apoptosis or programmed cell death. This antibody is produced in rabbits and recognizes the cleaved form of caspase-3, which is a widely used marker for the identification of cells undergoing apoptosis.

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20 protocols using rabbit polyclonal anti cleaved caspase 3

Immunohistochemical Profiling of Cell Markers

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IHC stain was performed on 4 μm thick tissue sections using the following antibodies and dilutions: mouse monoclonal anti-Ki-67, clone MIB-1 (DAKO/Agilent, Santa Clara, CA, USA; cat# M7240) with a dilution of 1:100, rabbit polyclonal anti-cleaved caspase-3 (Cell Signaling, Danvers, MA, USA; cat# 9661) with a dilution of 1:100, rabbit monoclonal anti-pS6 (S235/236), clone D57.2.2E and rabbit polyclonal anti-pS6 (S240/244) (Cell Signaling, Danvers, MA, USA, cat# 4858 and cat# 2215) both pS6 antibodies were used with a dilution of 1:100. All stains were evaluated by a pathologist (CT). For Ki-67 and cleaved caspase-3 the fraction of positive cells was estimated (percentage) and for the pS6 antibodies the percentage of positivity and the staining intensity was estimated resulting in an H-score (0–300).

Owusu-Brackett N., Evans K.W., Akcakanat A., Yuca E., Tapia C., Rizvi Y.Q., Dumbrava E.I., Janku F, & Meric-Bernstam F. (2019). TAK228 enhances antitumor activity of eribulin in triple negative breast cancer. Oncotarget, 10(49), 5011-5019.

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Multiparametric Fluorescent Imaging Assay

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Tetramethylrhodamine, methyl ester (TMRM), Hoechst 33342, LysoTracker® Red DND-99, LysoTracker® Green DND-26, 5-(and-6)-chloromethyl-2′,7’-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H₂DCFDA), CellLight® Nucleus-GFP, Alexa Fluor® 633 phalloidin, goat anti-mouse Alexa Fluor® 633, goat anti-rabbit Alexa Fluor® 633, goat anti-mouse Alexa Fluor® 488, and goat anti-rabbit Alexa Fluor® 488 were purchased from Molecular Probes (Thermo Fisher Scientific). Resazurin, pyrimethamine, methotrexate, aminopterin, and most of the drugs were purchased from Sigma. The rabbit polyclonal anti-AFP (Dako, Denmark A/S, Denmark), mouse monoclonal anti-CHALV1 (Abcam, CSP, Cambridge, England), rabbit polyclonal anti-cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA), mouse monoclonal anti-phospho-histone H2AX (γ-H2AX) (Millipore, Bedford, MA, USA), and rabbit polyclonal anti-cathepsin B (EMD/Calbiochem, San Diego, CA, USA) antibodies were purchased from each of the indicated companies.

Jang J.W., Song Y., Kim K.M., Kim J.S., Choi E.K., Kim J, & Seo H. (2016). Hepatocellular carcinoma-targeted drug discovery through image-based phenotypic screening in co-cultures of HCC cells with hepatocytes. BMC Cancer, 16, 810.

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Western Blot Analysis of Cellular Proteins

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Western blot analysis was carried out as previously described.19 The following antibodies were used: rabbit polyclonal anti‐BMI‐1 (Proteintech, Chicago, IL, USA), mouse monoclonal anti‐ β‐actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit polyclonal anti‐cleaved caspase‐3 (Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti‐ubiquitinated histone H2A (uH2A; Cell Signaling Technology), mouse monoclonal anti‐histone H2A (Cell Signaling Technology), and rabbit polyclonal anti‐poly (ADP‐ribose) polymerase (PARP) (Cell Signaling Technology).

Nishida Y., Maeda A., Chachad D., Ishizawa J., Qiu Y.H., Kornblau S.M., Kimura S., Andreeff M, & Kojima K. (2015). Preclinical activity of the novel B‐cell‐specific Moloney murine leukemia virus integration site 1 inhibitor PTC‐209 in acute myeloid leukemia: Implications for leukemia therapy. Cancer Science, 106(12), 1705-1713.

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Western Blot Protein Analysis

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Tissue and cell samples were homogenized in lysis buffer,^{47 (link)} then separated by 10 or 12% SDS-PAGE under reducing conditions and transferred to PVDF membranes (Millipore, Bedford, MA, USA), blocked with 5% skimmed milk in PBS/0.5% v/v Tween 20 for 1 h, and washed with PBS/Tween. Primary antibody was goat polyclonal anti-BASP1 (1 : 500, Santa Cruz, Santa Cruz, CA, USA), rabbit polyclonal anti-cleaved caspase-3 (1 : 1000; Cell Signaling, Hertfordshire, UK), rabbit monoclonal anti-cleaved PARP (1 : 1000, Abcam, Cambridge, UK), mouse anti-tubulin monoclonal antibody (1 : 5000, Sigma) or mouse GAPDH (Millipore, Billerica) followed by incubation with horseradish peroxidase-conjugated secondary antibody (1 : 2000, Amersham, Aylesbury, UK). Blots were developed with the enhanced chemiluminescence method following the manufacturer's instructions (Amersham). Levels of expression were corrected for minor differences in loading.

Sanchez-Niño M.D., Fernandez-Fernandez B., Perez-Gomez M.V., Poveda J., Sanz A.B., Cannata-Ortiz P., Ruiz-Ortega M., Egido J., Selgas R, & Ortiz A. (2015). Albumin-induced apoptosis of tubular cells is modulated by BASP1. Cell Death & Disease, 6(2), e1644-.

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Immunohistochemical Analysis of Apoptosis Markers

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Tissue sections were stained with rabbit polyclonal anti‐cleaved Caspase‐3 (Cell Signaling), rabbit monoclonal anti‐p16, rabbit polyclonal anti‐Bcl‐2, or rabbit monoclonal anti‐Bcl‐XL (all from Abcam) antibodies. The slides were blocked for endogenous peroxidase with 3% H2O2 and boiled for antigen retrieval in citrate buffer (pH 6). Sections were incubated with the appropriate secondary antibody from Vector Laboratories (goat anti‐rabbit IgG antibody (H + L), washed, and incubated with the VECTASTAIN®Elite ABC Peroxidase standard kit (Vector laboratories, Newark, NJ). After several washes, the chromogen 3,3′‐diaminobenzidine from the Peroxidase Substrate Kit (Vector Laboratories) was added to each slide. The slides were counterstained with Mayer's Hemalun (Merck). Lastly, the slides were mounted with glycerin mounting medium (Dako). Images were acquired using an Evos M5000 microscope.

Cobanoglu O., Delval L., Ferrari D., Deruyter L., Heumel S., Wolowczuk I., Hussein A., Menevse A.N., Bernard D., Beckhove P., Alves F, & Trottein F. (2023). Depletion of preexisting B‐cell lymphoma 2‐expressing senescent cells before vaccination impacts antigen‐specific antitumor immune responses in old mice. Aging Cell, 22(12), e14007.

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Comprehensive Antibody Panel for Protein Analysis

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We used the following antibodies: mouse monoclonal anti-COMMD1 (clone 3B3; Abcam; ab131597), rabbit polyclonal anti-COMMD1 (Proteintech Group; 11938-1-AP), mouse monoclonal anti-β-Actin (Sigma-Aldrich; A5441), rabbit polyclonal anti-Tubulin (Abcam; ab4047), rabbit polyclonal anti-Lamin A/C (Cell Signaling Technology; #2032), mouse monoclonal anti-Bcl2 (Santa Cruz Biotechnology; sc-509), rabbit monoclonal anti-PARP (Cell Signaling Technology; #9532), rabbit polyclonal anti-cleaved-caspase3 (Cell Signaling Technology; #9661), mouse monoclonal anti-phospho-Ser10-Histone H3 antibody, (Cell Signaling Technology; #9706), mouse monoclonal anti-XIAP (BD Biosciences, # 610716), rabbit polyclonal anti-BRCA1 (Cell Signaling Technology; #9010), mouse monoclonal anti-Hsp90 (AC88) (Enzo; ADI-SPA-830), rabbit monoclonal anti-phospho-Ser139-H2AX (Cell Signaling Technology; #9718), Alexa-488-conjugated polyclonal anti-mouse (Molecular Probes; A-11001), Alexa-647-conjugated polyclonal anti-rabbit (Molecular Probes, A-21244), HRP-conjugated polyclonal goat anti-rabbit IgG (H + L) (Bio-Rad; #170–6515), HRP-conjugated polyclonal goat anti-mouse IgG (H + L) (Bio-Rad; #170–6516).

Fedoseienko A., Wieringa H.W., Wisman G.B., Duiker E., Reyners A.K., Hofker M.H., van der Zee A.G., van de Sluis B, & van Vugt M.A. (2016). Nuclear COMMD1 Is Associated with Cisplatin Sensitivity in Ovarian Cancer. PLoS ONE, 11(10), e0165385.

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Autophagy and Apoptosis Regulation

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Histamine dyhydrochloride (C5H9N3.2HCL), 3-MA (3-methyl-3H-purin-6-amine), Z-VAD-fmk, Z-IETD-fmk, pyrilamine, cimetidine, and bafilomycin A1 were obtained from Sigma. Antibodies were obtained from the following sources: rabbit polyclonal anti-LC3 (Abcam), Rabbit polyclonal p62 antibody (Abcam), rabbit polyclonal anti-Histamine Receptor 1 (ABGENT), rabbit polyclonal anti-Histamine Receptor 2 (ABGENT), rabbit polyclonal anti-cleaved caspase-3 (Cell Signaling, 9661), mouse monoclonal anti-caspase-8 (Cell Signaling), rabbit polyclonal anti-Atg13 (Abcam), mouse monoclonal anti-FADD (Abcam), rabbit monoclonal anti-Beclin 1 (Abcam).

Ding S., Abudupataer M., Zhou Z., Chen J., Li H., Xu L., Zhang W., Zhang S., Zou Y., Hong T., Wang T.C., Yang X, & Ge J. (2018). Histamine deficiency aggravates cardiac injury through miR-206/216b-Atg13 axis-mediated autophagic-dependant apoptosis. Cell Death & Disease, 9(6), 694.

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Antibody-based Western Blotting Analysis

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Western blotting analysis was performed as the previous study.^{19 (link)} Antibodies used were as following: rabbit polyclonal anti-PARP, rabbit polyclonal anti-caspase-3, rabbit polyclonal anti-cleaved caspase-3, rabbit polyclonal anti-caspase-9, rabbit polyclonal anti-cleaved caspase-9, rabbit polyclonal anti-cyclin B1, mouse polyclonal anti-cdc2 and rabbit polyclonal anti-p-cdc2 (Tyr15) were purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse polyclonal anti-MPM2 and rabbit polyclonal anti-BICD2 were commercially available from Millipore Corporation (Bedford, MA, USA), and rabbit polyclonal anti-FOXM1 antibody was purchased from ABclonal Technology (Wuhan, China). Mouse polyclonal anti-β-actin was purchased from Sunshine Biotechnology Ltd. Goat polyclonal anti-rabbit IgG conjugated to HRP and goat polyclonal anti-mouse IgG conjugated to HRP (Cell Signaling Technology) were used as secondary antibodies, and enhanced chemiluminescence reagents (Millipore) was used for detection and exposed by Gel 2000 image analyzer (Bio-Rad, Richmond, CA, USA).

Li H., Sun L., Li H., Lv X., Semukunzi H., Li R., Yu J., Yuan S, & Lin S. (2017). DT-13 synergistically enhanced vinorelbine-mediated mitotic arrest through inhibition of FOXM1-BICD2 axis in non-small-cell lung cancer cells. Cell Death & Disease, 8(5), e2810-.

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Immunoblotting Protocol for CDK2, Cyclin E2, and Apoptosis Markers

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Immunoblotting was performed as decribed in51 (link). Primary antibodies used were mouse monoclonal anti-CDK2 (Santa Cruz Biotechnology, Dallas, Texas, USA), rabbit polyclonal anti-CDK2 (Abcam, Cambridge, UK), rabbit polyclonal anti-phospho-CDK2 (Cell Signaling Technology, Danvers, MA), rabbit polyclonal anti-cyclin E2 (Cell Signaling Technology), rabbit polyclonal anti-CDK4 (Cell Signaling Technology), rabbit polyclonal anti-p38 MAPK, rabbit polyclonal anti-phospho-p38 MAPK (Thr180/Tyr182), rabbit polyclonal anti-phopho-Akt (Ser473), rabbit polyclonal anti-cleaved caspase-3 (Cell Signaling Technology), mouse monoclonal anti-Pan Akt (R&D Systems, Minneapolis, MN), rabbit polyclonal anti-Bax and rabbit polyclonal anti-Bcl2 (Abcam) and mouse monoclonal anti-tubulin IgGs (Sigma-Aldrich). Alexa Fluor 680 (Invitrogen) and IRDye 800 (LI-COR, Lincoln, NE) donkey anti-rabbit, anti-goat or anti-mouse IgGs were used as secondary antibodies. Detection and quantification was performed with an Odyssey Infrared Imager (LI-COR). Full Western blots with antibodies against CDK2 and phospho-CDK2 are shown in Supplementary Figure S5.

Saurus P., Kuusela S., Dumont V., Lehtonen E., Fogarty C.L., Lassenius M.I., Forsblom C., Lehto M., Saleem M.A., Groop P.H, & Lehtonen S. (2016). Cyclin-dependent kinase 2 protects podocytes from apoptosis. Scientific Reports, 6, 21664.

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Antibodies Used in Cell Signaling

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The following antibodies were used: mouse monoclonal anti-citron (#611377; Transduction Laboratories, BD Biosciences, Franklin Lakes, NJ, USA), mouse monoclonal anti-vinculin (#V9131), mouse monoclonal anti-αTubulin (#T5168) from Sigma-Aldrich; rabbit polyclonal anti-cleaved Caspase 3 (#9661S), anti-TP53 phospho Ser15 (#9284S), anti-γH2AX (S139; 20E3; #2577), anti FLT3 (8F2 #3462) and anti-INCENP (P240, #2807) from Cell Signaling Technology (Danvers, DA, USA); rabbit polyclonal anti-TP73 (#ab14430) and rabbit monoclonal anti-53BP1 (#ab36823) from Abcam (Cambridge, UK); rabbit monoclonal anti-RAD51 (#sc-8349) and rabbit polyclonal anti p21 (#sc-756) from Santa Cruz (Dallas, TX, USA), rabbit polyclonal anti-pTSS INCENP (a kind gift of M.A. Lampson) (28 (link)).

Pallavicini G., Iegiani G., Parolisi R., Ferraro A., Garello F., Bitonto V., Terreno E., Gai M, & Di Cunto F. (2023). Lestaurtinib inhibits Citron kinase activity and medulloblastoma growth through induction of DNA damage, apoptosis and cytokinesis failure. Frontiers in Oncology, 13, 1202585.

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