Total protein from JEG3 and JEG3 tumors from different treatment groups was run in PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel and transferred to a PVDF (polyvinylidene fluoride) membrane and probed with the following primary antibodies, for 12 h, at 4 °C: caspase-3, caspase-9, p53, bax, bcl-2, AIF, cIAP 1 and cIAP 2 (Abcam Inc., Cambridge, MA, USA, 1:1000), X-linked inhibitor of apoptosis protein (XIAP) (Santa Cruz Biotechnology Inc., USA, 1:1000), poly (ADP-ribose)-polymerases (PARPs), and β-actin (Abcam Inc., Cambridge, MA, USA, 1:1000). Then, the blots were incubated with HRP (horseradish peroxidase)-conjugated goat anti-rabbit secondary antibody for 1 h and washed. Following detection with FluorChem E Chemiluminescent Western Blot Imaging System (Cell Biosciences, Santa Clara, CA, USA), the blots were quantified by densitometry analysis, using Image J software (National Institutes of Health, Bethesda, MD, USA). All Western blot data shown are representative of at least three independent experiments.
Ciap1
Manufactured by Abcam
Sourced in United States, United Kingdom
CIAP1 is a protein encoded by the BIRC2 gene. It functions as an inhibitor of apoptosis, regulating cell death and survival pathways.
Automatically generated - may contain errors
Lab products found in correlation
Ciap2, by Abcam (5 mentions)
Caspase 9, by Abcam (2 mentions)
β actin, by Abcam (2 mentions)
Gapdh, by Abcam (2 mentions)
Bcl 2, by Abcam (1 mentions)
Caspase 3, by Abcam (1 mentions)
Bcl2 associated x (bax), by Abcam (1 mentions)
X linked inhibitor of apoptosis protein xiap, by Santa Cruz Biotechnology (1 mentions)
Fluorchem e chemiluminescent western blot imaging system, by Cell Biosciences (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Odyssey infrared imager, by LI COR (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Infrared dye labeled secondary antibody, by LI COR (1 mentions)
Caspase 8, by Cell Signaling Technology (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Caspase 9, by Cell Signaling Technology (1 mentions)
Ciap2, by BD (1 mentions)
Nf κb p65, by Abcam (1 mentions)
Curcumin, by Merck Group (1 mentions)
Cisplatin, by Merck Group (1 mentions)
6 protocols using ciap1
1
Western Blot Analysis of Apoptosis Markers
2
Western Blot Protein Analysis Protocol
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Cell pellets were lysed in RIPA lysis buffer and the protein content was determined using a DC protein assay kit (Bio-Rad Laboratories). The proteins were run on electrophoresis gels and transferred to nitrocellulose membranes (GE Osmonics Labstore) as described previously [50 (link)]. Membranes were blocked for 1 hr in blocking buffer, incubated with primary antibodies against XIAP (BD Biosciences, 610762; as per company, this antibody detects two bands and the lower band is specific for XIAP, as indicated by arrows in the figures); cIAP1 (Abcam, ab2399); cIAP2 (Epitomics, S2700; as per company, this antibody detects two bands and the upper band is specific for cIAP2, as indicated by arrows in the figures); Smac (BD Biosciences, 612246); caspase 3, (Cell Signaling, 9665) caspase 8 (Cell Signaling 9746), and caspase 9 (Cell Signaling, 9502); polyclonal antibody to Mcl-1 or Bcl-xL; and mouse monoclonal antibody to Bcl-2 (Santa Cruz, CA) or to GAPDH (Abcam, Cambridge, MA). The antibody to poly(ADP-ribose) polymerase was from BIOMOL International (Plymouth Meeting, PA). Following washing with PBST, membranes were incubated for 1 hr with infrared dye-labeled secondary antibodies (LI-COR Biosciences), scanned, and visualized using a LI-COR Odyssey infrared imager.
3
Cisplatin and Curcumin Modulate NF-κB Signaling
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Cisplatin and curcumin were purchased from Sigma, the cell counting kit‐8 was purchased from DOjinDO (CCK‐8, DOjinDO Laboratories), and the miRNeasy mini‐kit was purchased from Qiagen. MiR‐192 mimics and miR‐192 inhibitor were obtained from GenePharma. Lipofectamine 2000 transfection reagent was purchased from Life technologies. Monoclonal antibody specific to NKRF, p‐κB, IκB, NF‐κB p65, PARP‐1, cIAP1, cIAP2, Bcl‐xl and XIAP were purchased from Abcam, cell signaling technology (Danvers) and Santa Cruz, respectively. pNFκB‐luc was purchased from Beyotime, pMIR‐GLO dual‐luciferase miRNA target report vector and dual‐luciferase reporter assay system were purchased from Promega.
4
Western Blot Analysis of Apoptosis Regulators
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The antibodies against A20, Survivin, c-IAP-1, c-IAP-2 and GAPDH were purchased from Abcam cooperation. Anti-rabbit or anti-mouse IgG antibodies conjugated with horseradish peroxidase (HRP) were purchased from Sigma (St. Louis, MO, USA). Cells, which were transfected with plasmids, were then harvested for western blots. Total protein samples were extracted from HCC cells and performed by SDS-PAGE, and trans-printed to poly-vinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked and then incubated with primary antibodies against A20 (1:2000 dilution), Survivin (1:500), c-IAP-1 (1:500), c-IAP-2 (1:500) or GAPDH (1:5000). The blots were then incubated with the HRP-conjugated secondary antibodies (1:5000). At last, blots were developed with enhanced chemiluminescence reagents (Pierce, Rockford, IL, USA) by X-ray films.
5
Western Blot Analysis of Apoptotic Markers
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25 µg of proteins were separated by SDS-PAGE on a 10% polyacrylamide gel. Proteins were transferred to a polyvinylidene difluoride membrane. Membranes were saturated for 1 h in tris-buffered saline (TBS) (50 mM Tris, 150 mM NaCl)/0.1% Tween-20/5% milk and incubated overnight at 4 °C with a rabbit primary antibody directed against: PARP (polyclonal, 1:1000, Cell Signaling), XIAP (monoclonal, 1:1000, Cell Signaling), cIAP1 (1:1000, Abcam) or caspase 9 (1:1000, Abcam). Membranes were washed three times with TBS/0.1% Tween 20 (TT) then incubated for 1 h 30 with a secondary antibody anti-rabbit (1:2000, Cell Signaling) coupled with HRP (horse raddish peroxydase). Membranes were washed three times with TT. Immunocomplexes were revealed by enhanced chemiluminescence (GE Healthcare, Amersham) and visualized with a photon camera (ChemiDoc, BioRad). Picture analyses were realized with Image Lab.
6
Apoptosis Signaling Pathway Analysis
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Western blot and co-immunoprecipitation assay were performed as described previously [20 (link)] using the following antibodies: Survivin, anti-FADD, pro-caspase-8 (Santa Cruz Biotechnology, CA, USA), XIAP (Epitomics, CA, USA), cleaved Caspase-8, TNFα, TRAF2, TRADD (Cell Signaling Technology). cIAP1, cIAP2, RIP1, TNFR1 and cleaved Caspase-3 and β-Actin (Abcam, Cambridge, UK) was used as a loading control. Proteins were detected using ECL detection (Thermo scientific, MA, USA). All western blot data shown are representative of at least three independent experiments.
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