Briefly, total proteins from cell lysates were subjected to SDS-PAGE followed by transfer onto polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA). The membranes were probed with Abs specific for c-IAP-1, c-IAP-2, Ca2+/calmodulin-dependent protein kinase II (CaMK-II), Bid, full-length caspase-8, Bax, TRAF-1, TRAF-2, Mcl-1, BcL-XL, and GAPDH (all from Cell Signaling Technology, Danvers, MA), cleaved caspase-8 (Santa Cruz Biotechnology, Santa Cruz, CA) followed by donkey anti-rabbit secondary polyclonal Abs conjugated to HRP (Amersham Bioscience, Montreal, Quebec). All immunoblots were visualized by ECL (Amersham Bioscience), as described previously (10 (link)).
Cleaved caspase 8
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Cleaved caspase-8 is an enzyme that plays a central role in the initiation of apoptosis, or programmed cell death. It is a critical component of the apoptotic signaling cascade, responsible for the cleavage and activation of downstream effector caspases, leading to the dismantling of the cell.
Automatically generated - may contain errors
Lab products found in correlation
Cleaved caspase 3, by Santa Cruz Biotechnology (5 mentions)
Cleaved parp, by Santa Cruz Biotechnology (3 mentions)
Bcl 2, by Santa Cruz Biotechnology (3 mentions)
β actin, by Abcam (2 mentions)
Caspase 3, by Santa Cruz Biotechnology (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (2 mentions)
Cleaved caspase 9, by Santa Cruz Biotechnology (2 mentions)
Traf1, by Cell Signaling Technology (1 mentions)
Traf2, by Cell Signaling Technology (1 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Bcl xl, by Cell Signaling Technology (1 mentions)
Mcl 1, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Ecl plus system, by Cytiva (1 mentions)
Cyclin d1, by BD (1 mentions)
P p70s6k, by Cell Signaling Technology (1 mentions)
α tubulin, by Abcam (1 mentions)
9 protocols using cleaved caspase 8
1
Western Blot Analysis of Apoptosis Regulators
2
Western Blot Analysis of Cell Signaling
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The cells were washed three times with ice-cold PBS before lysis, and were lysed with a buffer containing 1% Triton X-100, 1% Nonidet P-40 (NP-40), as well as protease and phosphatase inhibitor cocktails (GenDEPOT, Barker, TX). Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto the polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA). Immunostaining was performed by incubating the blots with primary antibodies, followed by horseradish peroxidase (HRP)-conjugated secondary antibody and detected with an enhanced chemiluminescence (ECL) plus system (Amersham Biosciences, Piscataway, NJ). The primary mouse antibodies against the following factors were used: p27 (Cell Signaling Technologies, Danvers, MA), cyclin D1, hypoxia-inducible factor-1α (HIF-1α, BD Biosciences, SanJose, CA), VEGF (Novus Biologicals, Littleton, CO) and β-actin (Abcam, Cambridge, MA). The primary rabbit polyclonal antibodies against the following proteins were also used: cleaved caspase-3, poly (ADP-ribose) polymerase protein (PARP), p-Akt, Akt, p-mTOR, mTOR, p-p70S6K, p70S6K, p-4E-BP1, 4E-BP1 (Cell Signaling Technologies), cleaved caspase-8, procasepase-9 (Santa Cruz Biotechnology, Santa Cruz, CA), and α-tubulin (Abcam). The secondary antibodies were purchased from Amersham Biosciences.
3
Cell Line Characterization and Immunoblot Analysis
4
Western Blot Analysis of Biochemical Markers
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The following materials were obtained from commercial sources: all chemical reagents from Sigma (St. Louis, MO). Antibodies for Western blotting were purchased as follows: β-Actin, NF-κB p65, NF-κB p50, Lamin B, 15-hydroxyprostaglandin dehydrogenase (15-PGDH), NACHT, LRP and PYD domains-containing protein 3 (NLRP3), IL-18, IL-1β, caspase-3, heme oxygenase-1 (HO-1), β-catenin, Bcl-2, Bax, cleaved PARP, cleaved caspase-3, and cleaved caspase-8 from Santa Cruz Biotechnology (Santa Cruz, CA), iNOS from BD Biosciences (San Jose, CA), COX-2 from Thermo Scientific (Seoul, Korea), HSP70, HSP60, HSP27, NQO-1, GST(pi), glutathione peroxidase (GPX), ERK1/2, and p-ERK1/2 from Abcam (Cambridge, MA). Horseradish peroxidase-conjugated anti-rat/rabbit/mouse IgG was purchased from Thermo Scientific Pierce (Rockford, IL).
5
Western Blot Analysis of Apoptotic Signaling
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Cells were harvested and lysed with RIPA buffer (Sigma-Aldrich) containing protease and phosphatase inhibitors (Roche, Switzerland). Protein lysates were separated on a 10–12% SDS-PAGE gel, transferred to polyvinylidene difluoride (PVDF) membranes, and blocked with 5% (w/v) skim milk in Tris-buffered saline (TBS) supplemented with 0.1% Tween-20 (TBS-Tw) for 1 h at room temperature. The PVDF membranes were then incubated with primary antibodies (Cell Signaling Technology, USA: total/cleaved-PARP total/cleaved-caspase-3, total/cleaved-caspase-7, cleaved-caspase-8, total/cleaved-caspase-9, total/phosphorylated-eIF2α; Santa Cruz Biotechnology: G3BP1, TIA-1, GFP, and eIF3η) at 4°C overnight, followed by HRP-conjugated anti-rabbit or anti-mouse IgG secondary antibodies for 1 h at room temperature. Tubulin or β-actin (Abgent, USA) antibody was used as a loading control. The membranes were incubated with Pierce ECL Western Blotting Substrate (ECL Solution Kit; Thermo Fisher Scientific). Band signals were displayed in a Fusion Solo Imaging System (Vilber Lourmat, France). Band intensity measurement was performed by ImageJ software.
6
Linalool Induces Apoptosis in 22Rv1 Cells
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The 22Rv1 cells (1×106) were seeded in a 25-cm2 flask and treated with linalool (2.5 mM) for 24 h at 37°C. After the treatment, the cells were collected and lysed with RIPA buffer on ice for 30 min. The protein concentration was determined by a BCA kit (Beyotime Institute of Biotechnology). The proteins (30 µg per lane) were separated on a 10% SDS-PAGE gel and electrotransferred onto a PVDF membrane. The membrane was blocked with 5% bovine serum albumin at room temperature for 1 h, and then immunoblotted with antibodies against caspase-3, cleaved caspase-3 (cat. nos. 9665 and 9664, respectively; both 1:1,000; Cell Signaling Technology, Inc.), caspase-8 (cat. no. SC56070; 1:500 dilution; Santa Cruz Biotechnology, Inc.), cleaved caspase-8 (cat. no. 9748; 1:1,000; Cell Signaling Technology, Inc.), caspase-9, cleaved caspase-9, DR4, DR5 (cat. nos. ab32539, ab2324, ab8414 and ab8416, respectively; all 1:1,000; Abcam), p53 (cat. no. AF0879; 1:1,000; Affinity Biosciences, Inc.), Bcl-2 (cat. no. ab59348; 1:1,000; Abcam), Bax, and β-actin (cat. nos. GB11007 and GB12001, respectively; 1:300 and 1:3,000, respectively; Wuhan Servicebio Technology, Co., Ltd.). The BeyoECL Plus kit (Beyotime Institute of Biotechnology) was used for visualization. The bands were analyzed by AlphaEaseFC 4.0 software (Genetic Technologies, Inc.).
7
Apoptosis Signaling Pathway Analysis
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Cellular protein was extracted by lysis buffer (Beyotime, Shanghai, China) and equal amounts of protein samples were separated on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel and blotted onto polyvinylidene difluoride membranes. After blocking within 5% skim milk for 1 h at room temperature, the membranes were incubated with primary antibodies: FASLG, Fas-associated via death domain (FADD), p-FADD, caspase-8, cleaved caspase-8, caspase-2, cleaved caspase-2, caspase-3, cleaved caspase-3 and GAPDH (1:1000; Santa Cruz, CA) at 4°C overnight. Then the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:1000; Santa Cruz, CA) for 1 h at room temperature. The immunoreactive protein bands were developed by enhanced chemiluminescence reagent (GE Healthcare, Little Chalfont, UK).
8
Apoptosis and Invasion Assays
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MTT, fetal bovine serum (FBS), DMSO, and L-Securinine were obtained from Sigma (St. Louis, MO, U.S.A.). Anti-Bax, Bcl-2, cleaved caspase-9, cleaved caspase-8, cleaved caspase-3, cytosolic cytochrome c, cleaved PARP, MMP-9, MMP-2, AGTR1, MEK1/2, p-MEK1/2, ERK1/2, p-ERK1/2, STAT3, p-STAT3, PAX2, p-PAX2, and VEGF antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, U.S.A.). Annexin V–fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit, Transwell chambers and Matrigel gel was from BD Biosciences (San Jose, CA, U.S.A.).
9
Cytoprotective Effects of Morin
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Dulbecco’s modified eagle’s media (DMEM), antibiotic-antimycotic mix, MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide), 2′,7′-dichlorofluorescin-diacetate (DCFH-DA), Rhodamine 123 (Rh123), Hoechst 33342, morin and glutaraldehyde were purchased from Sigma–Aldrich Chemicals (St Louis, MO, United States). Fetal bovine serum (FBS) was purchased from Gibco-BRL (Auckland, New Zealand). Annexin V – FITC assay kit (600300) was purchased from Cayman chemicals. Glutathione colorimetric assay kit (K261-100) and catalase activity assay kit (K773-100) were purchased from Biovision. BCA protein assay kit was procured from Pierce Biotechnology, Rockford, IL, United States. Caspase 3 specific inhibitor, z-DEVAD-fmk was purchased from R&D Systems (Minneapolis, MN, United States). Primary antibodies (β actin, cleaved PARP, cleaved caspase 3, cleaved caspase 8, cleaved caspase 9, Bcl 2, Bax and Glut 1) and corresponding secondary antibodies for western blot analysis were purchased from Santa Cruz Biotechnology, United States. Clarity Western ECL substrate was purchased from Bio-rad, United States. 2-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) amino-2-deoxy-D -glucose (2-NBDG) was obtained from Molecular Probe (Invitrogen Life Technologies, Carlsbad, CA, United States). All other chemicals used were of the standard analytical grade.
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