Anti caspase 3

Manufactured by Cell Signaling Technology

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Anti-caspase-3 is a laboratory reagent used to detect and quantify the presence of caspase-3, a key enzyme involved in apoptosis (programmed cell death). It is a specific antibody that binds to caspase-3, allowing its identification and measurement in cell and tissue samples.

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Lab products found in correlation

744 protocols using anti caspase 3

Protein Expression Analysis via Western Blot

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Cells were lysed in RIPA-SDS buffer, after which the proteins were resolved by
SDS-PAGE, transferred onto PVDF membranes, and probed with the relevant
antibodies as previously described9 (link). The following antibodies
were used for the experiments: anti-actin (Sigma); anti-SESN2 (Proteintech),
anti-NRF2, anti-CHOP and anti-p53 (Santa Cruz); and anti-phospho(T389)-p70S6K,
anti-phospho(S235/236)-S6, anti-phospho(S65)-4E-BP1, anti-phospho
IF2α(S51), anti-phospho(T172)-AMPKα,
anti-phospho(S79)-acetyl-CoA carboxylase, anti-phospho(S9)-GSK3β,
anti-phospho(T1462), anti-4E-BP1, anti-S6, anti-TSC2, anti-AMPKα,
anti-ATF4, anti-Bip, anti-p62, anti-LC3, anti-PARP, anti-PARP(cleaved),
anti-caspase3, and anti-caspase3(cleaved) (Cell Signaling Inc). Sesn1 antibodies
were previously described11 (link).

Ding B., Parmigiani A., Divakaruni A.S., Archer K., Murphy A.N, & Budanov A.V. (2016). Sestrin2 is induced by glucose starvation via the unfolded protein response and protects cells from non-canonical necroptotic cell death. Scientific Reports, 6, 22538.

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Apoptosis Induction by Targeted Doxorubicin

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MCF-7/Adr cells were cultured for 12 h and then treated with free DOX, DOX nanoparticles, targeted DOX nanoparticles or functional DOX nanoparticles. Controls were performed by adding blank medium. The final concentration of DOX was 2 μM. After 12 h of incubation, the cells were harvested, lysed, and analyzed by western blotting. The following antibodies were used: anti-caspase-8, anti-caspase-9, anti-caspase-3, and anti-PARP (all from Cell Signaling, Beverly, MA, USA) [27 (link)].

Wang H., Yin H., Yan F., Sun M., Du L., Peng W., Li Q., Feng Y, & Zhou Y. (2014). Folate-mediated mitochondrial targeting with doxorubicin-polyrotaxane nanoparticles overcomes multidrug resistance. Oncotarget, 6(5), 2827-2842.

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Ni(SalPipNONO) Mechanism in A549 Cells

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Sub-confluent A549 were seeded in 6 cm diameter Petri dishes. After adherence, cells were treated for the indicated times with Ni(SalPipNONO) (0.5 mM) and specific pathway inhibitors. Protein extraction and Western blot were performed as previously described [58 (link), 61 (link)]. Electrophoresis (50 μg of protein/sample) was carried out in 4–12% Bis-Tris Gels (Life Technologies, Carlsbad, CA, USA). Proteins were then blotted onto nitrocellulose membranes, incubated overnight with primary antibodies [Anti-cytochrome c, anti-phospho-ERK1/2, anti-caspase- 3, anti-ERK antibodies from Cell Signaling (Celbio, Milan, Italy); anti-p53 antibody from Santa Cruz Biotechnology Inc (Dallas TX, USA); anti-COX-2 from Cayman Chemical (Ann Arbor, MI, USA); anti-HIF-1α from BD Biosciences (San Jose, CA, USA); anti-VEGF from Merck KGaA (Darmstadt, Germany)] and then detected by enhanced chemiluminescence system (Biorad, Hercules, CA, USA). Results were normalized to those obtained by using an antibody against beta actin from Merck KGaA (Darmstadt, Germany) or total ERK1/2 (Cell Signaling, Celbio, Milan, Italy), when indicated.

Ciccone V., Monti M., Monzani E., Casella L, & Morbidelli L. (2018). The metal-nonoate Ni(SalPipNONO) inhibits in vitro tumor growth, invasiveness and angiogenesis. Oncotarget, 9(17), 13353-13365.

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Apoptosis and Autophagy Regulation in PBDE-47 Exposure

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The following antibodies were used: anti-PARP (Cell Signaling Technology, 9542), anti-caspase-3 (9661, Cell Signaling Technology, USA), anti-autophagy-related protein 7 (ATG7) (ab133528, Abcam, USA), anti-LC3 (14600-1-AP, Proteintech, USA), anti-p62 (ab56416, Abcam, USA), anti-GAPDH (60004-1-Ig, Proteintech, USA). The following chemical regents were used: PBDE-47 (purity 99.5%, GC/MS) (BDE-047N-3G, AccuStandard Corp, USA), Wortmannin (WM) (S2758, Selleck Chemicals, USA), Rapamycin (RAP) (R5000, Shanghai Haoran, China), Ac-DEVD-CHO (DEVD) (C1206-10 mM, Beyotime Institute of Biotechnology, China). All other chemical regents were analytical grade purchased from credible supplier or as described in the relevant methods.

Li P., Ma R., Dong L., Liu L., Zhou G., Tian Z., Zhao Q., Xia T., Zhang S, & Wang A. (2019). Autophagy impairment contributes to PBDE-47-induced developmental neurotoxicity and its relationship with apoptosis. Theranostics, 9(15), 4375-4390.

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Western Blot Analysis of Apoptosis Markers

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Sixty microgram of the cell extract was loaded onto an 8% polyacrylamide gel, separated by electrophoresis, and subsequently transferred onto a nitrocellulose membrane. The membrane was blocked (PBS 8% skimmed milk) at room temperature for 2 h and then incubated at 4 °C overnight with primary anti‐Apaf1 monoclonal antibody (AdipoGen, San Diego, CA, USA; AG‐20T‐0134‐c100), anti‐luciferase (Abcam, Cambridge, UK; ab21176), anti‐caspase‐9 (Cell Signaling Technology, Danvers, MA, USA; 9502), anti‐caspase‐3 (Cell Signaling Technology; 9662), cytochrome c (BD Pharmingen, San Diego, CA, USA; 556433), or actin (Proteintech, Rosemont, IL, USA; 66009‐1‐Ig), all diluted 1 : 1000. The membrane was washed with PBS containing 0.05% Tween‐20 (PBST) before being incubated with secondary antibody diluted 1 : 10 000: goat anti‐rat (Li‐COR, 925‐32219), goat anti‐rabbit (Li‐COR, 926‐32211), and goat anti‐mouse (Li‐COR, 926‐68020) in PBST. The membrane was then scanned using a LI‐COR system.

Tashakor A., H‐Dehkordi M., O'Connell E., Gomez Ganau S., Gozalbes R., Eriksson L.A., Hosseinkhani S, & Fearnhead H.O. (2019). A new split‐luciferase complementation assay identifies pentachlorophenol as an inhibitor of apoptosome formation. FEBS Open Bio, 9(7), 1194-1203.

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Western Blotting Analysis of Apoptosis Markers

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Cell lysates were prepared by resuspending cell pellets in 1× Laemmli sample buffer containing 5% β-mercaptoethanol. Protein from lung tissues were extracted using IPH lysis buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 5 mM EDTA, 0.5% NP40) containing protease inhibitor cocktail (Roche). The protein lysates were separated by SDS–PAGE and then electrotransferred to nitrocellulose membranes (Millipore Corp., Bradford, MA, USA). Detection of specific proteins was carried out with enhanced chemiluminescence reagents following the manufacturer’s instructions (P90720, Millipore Corporation, MA, USA). The primary antibodies used for western blotting were as follows: anti-Caspase-9 (#9508), anti-Caspase-8 (#9746), anti-Caspase-7 (#8438), anti-Caspase-3 (#9664), anti-Bim (#2933), anti-Bak (#3814), anti-Bcl-xL (#2762), anti-p-Jak2 (#3771), anti-Jak2 (#3230), and anti-p-STAT3^Y705 (#9145), which were purchased from Cell Signaling Technology. Anti-CDK2 (sc-6248), anti-p53 (sc-126), anti-STAT3 (sc-8019), anti-PARP-1 (sc-74470), anti-Cyclin D1 (sc-8396), and anti-β-actin (sc-47778) antibodies were purchased from Santa Cruz Biotechnology. An anti-p21 (ab7960), anti-p16 (ab108349), anti-IL-6 (ab6672), and anti-TNF-α (ab6671) antibodies were purchased from Abcam. Densitometry analyses were performed using ImageJ software (National Institutes of Health, NIH).

Cho H.J., Hwang J.A., Yang E.J., Kim E.C., Kim J.R., Kim S.Y., Kim Y.Z., Park S.C, & Lee Y.S. (2022). Nintedanib induces senolytic effect via STAT3 inhibition. Cell Death & Disease, 13(9), 760.

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Antibody and Chemical Sources for Cell Signaling

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The antibodies and chemicals were obtained from the following resources; anti-PARP (#556362), anti-XIAP (#610716), anti-FADD (#610399), anti-RIP1 (#610459), anti-p53 (#554147) and anti-p21 (#556430) antibodies (BD Biosciences, San Diego, CA, USA); anti-caspase-3 (#9662), anti-caspase-8 (#9746), anti-caspase-9 (#9508) and anti-Bid (#2002) antibodies (Cell signalling Technology, Beverly, MA, USA); anti-caspase-10 (M059-3) antibody (MBL, WOBURN, MA, USA); anti-Bcl-X_S/L (sc-271121), anti-survivin (sc-17779), anti-TRAF2 (sc-876), anti-GFP (sc-9996) and anti-HA (sc-805) antibodies (Santa Cruz, CA, USA); anti-c-FLIP (ALX-804-961) antibody (Enzo Life Sciences, Farmingdale, NY, USA); anti-cIAP1/2 (#07-759) antibody (Upstate Biotech, Waltham, MA, USA); anti-DR5 (#ab181846) antibody (Abcam, Cambridge, UK); anti-DR4 (NB100-56528) antibody (Novus, Centennial, CO, USA); anti-TurboGFP (PA5-22688) antibody (Thermo scientific, Waltham, Massachusetts, USA); anti-actin (A2066), anti-flag (F3165, 1:2,000 dilution) antibodies (Sigma-Aldrich, St. Louis, MO, USA). the pan caspase inhibitor Z-VAD-FMK, MG-132, TPCA-1 (Calbiochem, San Diego, CA, USA); recombinant TNF (R & D Systems, Minneapolis, MN, USA); recombinant human TRAIL/Apo2 ligand (Peprotech, Rocky Hill, NJ, USA).

Lee S.R., Quan K.T., Byun H.S., Park I., Kang K., Piao X., Ju E., Ro H., Na M, & Hur G.M. (2019). Accelerated degradation of cFLIPL and sensitization of the TRAIL DISC-mediated apoptotic cascade by pinoresinol, a lignan isolated from Rubia philippinensis. Scientific Reports, 9, 13505.

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Western Blot Analysis of Cell Cycle and Apoptosis

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30 μg of whole cell lysate was separated on 7.5–12% SDS-PAGE and Western blotting as previously described [12 (link)]. Cyclin-B was detected using anti-cyclin B (Sigma). Apoptotic markers including, caspase-3 and poly (ADP-ribose) polymerase (PARP) were detected using anti-caspase-3 (Cell Signaling) and anti-PARP (Cell Signaling) antibodies, respectively. Anti-β-actin antibody (Sigma), was used as the loading control.

Rattanapornsompong K., Khattiya J., Phannasil P., Phaonakrop N., Roytrakul S., Jitrapakdee S, & Akekawatchai C. (2021). Impaired G2/M cell cycle arrest induces apoptosis in pyruvate carboxylase knockdown MDA-MB-231 cells. Biochemistry and Biophysics Reports, 25, 100903.

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Protein Expression Analysis of DP T Thymocytes

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Total protein extracts were prepared from sorted DP T thymocyte samples and Western blotting analysis was conducted, as previously described (43 (link)), using anti-p21 antibody (C-19, sc-397, Santa Cruz), and anti-Caspase-3 or anti-cleaved Caspase-3 antibody (#9665 and #9661, respectively, both from Cell signaling). Anti-β-actin antibody (Sigma-Aldrich), was used to normalize protein expression levels. Densitometric analysis was performed using ImageStudio software (LI-COR Biosciences).

Grazioli P., Orlando A., Giordano N., Noce C., Peruzzi G., Scafetta G., Screpanti I, & Campese A.F. (2020). NF-κB1 Regulates Immune Environment and Outcome of Notch-Dependent T-Cell Acute Lymphoblastic Leukemia. Frontiers in Immunology, 11, 541.

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Molecular Mechanisms of Cell Cycle Regulation

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Imatinib, danusertib, volasertib and AZD1775 were purchased from Selleck Chemicals. For Western blotting, 10% acrylamide gels, running buffer (MOPS), transfer buffer and polyvinylidene difluoride (PVDF) transfer membrane were bought from Thermo Scientific. For immunofluorescence (IF), the FITC-conjugated anti-mouse IgG, the anti-rabbit conjugated with Alexa Fluor 568 antibodies and DAPI (6-diamidino-2-phenylindole) were purchased from Sigma-Aldrich.
The anti-cleaved-caspase 3 (Asp175), anti-cleaved-caspase 9 (Asp353), anti-BAX, anti-CHK1, anti-phospho-CHK1 (S317), anti-CHK2, anti-phospho-CHK2 (T68), anti-cyclin B1, anti-phospho-cyclin B1 (S133), anti CDC25C, anti-phospho-CDC25C (S198), anti-WEE1, anti-phospho-WEE1 (S642), anti-CDK1, anti-phospho-CDK1 (Y15), anti-phospho-H2AX (S139), anti-RAD51, anti- β-tubulin Alexa Fluor 555 Conjugate, anti-Aurora kinase A, anti-phospho-Aurora kinase A (T288), anti-PLK1, anti-phospho-PLK1 (T210), anti-PARP, anti-caspase-3 and anti-caspase-9 antibodies were purchased from Cell Signaling Technology. The anti-β-actin antibody used as loading control was purchased from Santa Cruz Biotechnology.

Mancini M., De Santis S., Monaldi C., Castagnetti F., Lonetti A., Bruno S., Dan E., Sinigaglia B., Rosti G., Cavo M., Gugliotta G, & Soverini S. (2022). Polo-like kinase-1, Aurora kinase A and WEE1 kinase are promising druggable targets in CML cells displaying BCR::ABL1-independent resistance to tyrosine kinase inhibitors. Frontiers in Oncology, 12, 901132.

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