The restriction endonucleases XbaI and NotI and T4 DNA ligase were purchased from New England Biolabs, Inc. (Ipswich, MA, USA). Paclitaxel was from LC Laboratories (Woburn, MA, USA). Trastuzumab (Herceptin) and Ado-trastuzumab emtansine (T-DM1, also known as Kadcyla) were obtained from University of Colorado Hospital pharmacy. Antibodies used for western blot assays were as follows: erbB3 (Ab7) (LabVision Corp. Fremont, CA, USA); P-erbB3, caspase-8 (1C12), and caspase-3 (8G10), P-MAPK, MAPK, P-Akt (S473), Akt, P-Src(Y416), Src, PARP (Cell Signaling Technology, Inc., Beverly, MA, USA); E2F1, Cyclin D1, p27kip1 (Santa Cruz Biotechnology, Inc. Dallas, TX, USA); and β-actin (Sigma Co., St. Louis, MO, USA). All other reagents were purchased from Sigma Co. unless otherwise specified.
Caspase 8 1c12
Manufactured by Cell Signaling Technology
Sourced in United States
Caspase-8 (1C12) is a mouse monoclonal antibody that recognizes the pro-form and large subunit of caspase-8, a key initiator caspase in the apoptosis signaling pathway. This antibody can be used for the detection of caspase-8 in various applications, such as Western blotting and immunoprecipitation.
Automatically generated - may contain errors
Lab products found in correlation
T4 dna ligase, by New England Biolabs (1 mentions)
β actin, by Merck Group (1 mentions)
P erbb3, by Cell Signaling Technology (1 mentions)
P27kip1, by Santa Cruz Biotechnology (1 mentions)
Paclitaxel, by LC Laboratories (1 mentions)
Xbai, by New England Biolabs (1 mentions)
Cyclin d1, by Santa Cruz Biotechnology (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Bip grp78, by Cell Signaling Technology (1 mentions)
Caspase 3 8g10, by Cell Signaling Technology (1 mentions)
Alpha tubulin, by Abcam (1 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
Xbp1s, by BioLegend (1 mentions)
Gabarap, by Proteintech (1 mentions)
Gabarapl1, by Abcam (1 mentions)
Gabarapl2, by Proteintech (1 mentions)
Fip200, by Proteintech (1 mentions)
Cleaved parp asp214 d64e10, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Gapdh d4c6r, by Cell Signaling Technology (1 mentions)
4 protocols using caspase 8 1c12
1
Molecular Mechanisms of Trastuzumab and T-DM1 Effects
2
Evaluation of Autophagy Markers in Tg-Treated Cells
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Cells were harvested after 30 h of treatment with Tg, unless otherwise stated. Preparation of whole-cell lysates, SDS-PAGE, and immunoblot analysis were performed as described previously [33 (link)]. The following antibodies were used: alpha-tubulin (Abcam, ab7291), ATG5 (Cell Signaling Technology 2630), ATF4 (Cell Signaling Technology 11815), Bip/Grp78 (Cell Signaling Technology 3177), Caspase-3 (8G10) (Cell Signaling Technology 9665), Caspase-8 (1C12) (Cell Signaling Technology 9746), CHOP (Cell Signaling Technology 2895), Cleaved PARP (Asp214) (D64E10) (Cell Signaling Technology 5625), DR5 (Cell Signaling Technology 8074), FADD (G-4) (Santa Cruz sc-271748), FIP200 (Proteintech 17250–1-AP, GABARAP (Proteintech PM037), GABARAPL1 (Abcam ab86497), GABARAPL2 (Proteintech PM038), p-JNK (Cell Signaling Technology 9251), JNK (Cell Signaling Technology 9252), LC3B (Cell Signaling Technology 2775), PERK (Cell Signaling Technology 5683), XBP1s (BioLegend 647502).
3
Western Blot Analysis of Caspase Activation
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Cellular protein in the transfected cells was extracted by RIPA lysis buffer (Beyotime, Shanghai, P.R. China). Protein samples were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes. The membranes were then blocked with 5% skim milk for 1 h at room temperature and were incubated with the special primary antibodies: caspase 8 (1C12), cleaved caspase 8 p18 (Asp387), or GAPDH (D4C6R) (all at a dilution of 1:1,000; Cell Signaling Technology, Beverly, MA, USA) at 4°C overnight. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Protein bands were developed using enhanced chemiluminescence and analyzed by ImageJ 1.49 (National Institutes of Health, Bethesda, MD, USA)19 (link).
4
Western Blot Analysis of Cell Signaling
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The cells were treated with vehicle or KB2 for 24 h and 48 h. The cells were lysed in whole-cell lysis buffer and western blotting was carried out as previously described (Bleloch et al., 2019 (link)). The primary antibodies used in this study were, rabbit polyclonal antibodies to phospho-histone H2A.X (Ser139) (#2577), cleaved caspase-3 (Asp175) (#9661), PARP (#9542), caspase-9 (#9502), LC3II (#2775), rabbit monoclonal antibody to β-Catenin (D10A8) (#8480), cleaved caspase-7 (Asp198) (D6H1) (#8438), mouse monoclonal antibodies to E-cadherin (4A2) (#14472), vimentin (R28) (#3932), Cyclin B1 (V152) (#4135), Caspase-8 (1C12) (#9746) from Cell Signaling Technology (Massachusetts, USA); mouse monoclonal antibody to p53 (DO-1) (sc-126), rabbit polyclonal antibodies to p21 (C-19) (sc-397), Cyclin A (H-432) (sc-751) from Santa Cruz Biotechnology (Texas, USA); rabbit polyclonal antibody to p38 MAP kinase (M0800) from Sigma Aldrich. After primary antibody incubation, membranes were incubated with goat anti-rabbit or goat anti-mouse HRP-conjugated secondary antibodies (Bio-Rad Laboratories, California, USA). p38 was used as a loading control. Densitometry readings were obtained using ImageJ and protein expression levels are represented as a ratio of protein of interest/p38 normalized to the vehicle control sample.
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