For immunoprecipitation, cytoplasm of BCSCs was probed with anti-XIAP (Cell Signaling Technologies, USA) overnight at 4°C. After that, protein A agarose beads were supplemented and maintained for two hours before they were collected by centrifugation. Subsequently, proteins on beads were released through boiling in sodium dodecyl sulfate (SDS) sample buffer. For western blot assay, proteins in cytoplasm of BCSCs were separated by 10% SDS-PAGE before they were transferred to nitrocellulose membranes. Subsequently, membranes were probed by anti-XIAP, anti-Smac/DIABLO, anti-caspase-3, anti-cleaved caspase-3, anti-cleaved PARP, anti-IgG HC and anti-GAPDH (Cell Signaling Technology) overnight at 4°C. Proper secondary antibodies were used to conjugate with the primary antibodies before the signals were detected by using enhanced chemilu-minescence detection kit (Pierce, USA).
Anti xiap
Manufactured by Cell Signaling Technology
Sourced in United States, Canada
Anti-XIAP is a primary antibody that specifically recognizes the X-linked Inhibitor of Apoptosis Protein (XIAP). XIAP is a key regulator of apoptosis, a form of programmed cell death. The antibody can be used to detect and analyze the expression of XIAP in various cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti survivin, by Cell Signaling Technology (13 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (11 mentions)
Anti cleaved parp, by Cell Signaling Technology (8 mentions)
Anti caspase 3, by Cell Signaling Technology (8 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (7 mentions)
Anti bim, by Cell Signaling Technology (7 mentions)
Anti parp, by Cell Signaling Technology (7 mentions)
Anti mcl 1, by Cell Signaling Technology (7 mentions)
Anti akt, by Cell Signaling Technology (6 mentions)
Anti cleaved caspase 8, by Cell Signaling Technology (6 mentions)
Anti bid, by Cell Signaling Technology (6 mentions)
Anti caspase 9, by Cell Signaling Technology (6 mentions)
Anti bcl xl, by Cell Signaling Technology (6 mentions)
Anti bcl 2, by Cell Signaling Technology (5 mentions)
Anti cleaved caspase 9, by Cell Signaling Technology (5 mentions)
Anti noxa, by Cell Signaling Technology (5 mentions)
Anti bcl xl, by Santa Cruz Biotechnology (5 mentions)
Anti puma, by Cell Signaling Technology (5 mentions)
Anti parp1, by Cell Signaling Technology (5 mentions)
Anti bax, by Santa Cruz Biotechnology (5 mentions)
46 protocols using anti xiap
1
XIAP Regulation in Breast Cancer Stem Cells
2
Comprehensive Protein Expression Analysis
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Western blot was performed as previously described (Lam et al., 2020 (link)). Specific primary antibodies [mouse monoclonal anti-human β-actin (Sigma-Aldrich); anti-PCNA, anti-PARP (Santa Cruz Biotechnology, Inc., Santa Cruz, California, United States of America); anti-PFKP, anti-LDHB, anti-Bcl-2, anti-survivin, anti-XIAP, anti-AKT, anti-CDK2, anti-CDK4, anti-CDK7, anti-Cyclin D2, H, anti-CDK4, anti-N-cadherin, anti-β-catenin (Cell Signaling Technology, Danvers, Massachusetts, United States of America); and corresponding horseradish peroxidase (HRP)-conjugated secondary (Cell Signaling Technology)] were purchased. An enhanced chemiluminescence (ECL) kit (GE Healthcare) was used to detect protein expression. Beta-actin was selected as a reference protein (Lam et al., 2020 (link)).
3
Metformin and TRAIL-Induced Apoptosis
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Metformin was purchased from Wako (Richmond, VA, USA). TRAIL (Recombinant human) was purchased from Millipore (Millipore, Darmstadt, Germany.) Protein G PLUS-Agarose, Anti-Bax, anti-Bcl-2, anti-Mcl-1(IP), anti-Ub and anti-Bcl-xL were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-XIAP, anti-phospho ERK, anti-ERK, anti-phospho JAK2, anti-JAK2, anti-phospho AMPK, anti-AMPK, anti-phospho mTOR, anti-mTOR, anti-phospho AKT, anti-AKT, anti-phsopho GSK3β, anti-GSK3β, anti-Noxa, anti-Puma, anti-Bim, anti-phospho Mcl-1, anti-Mcl-1(WB), anti-cleaved caspase-3, anti-cleaved caspase-8, anti-cleaved caspase-9, anti-phospho STAT3, anti-STAT3, and anti-PARP-1 were purchased from Cell Signaling (Beverly, MA, USA). Anti-actin antibody was purchased from Sigma (Sigma, St. Louis, MO). Anti-Mule antibody was purchased from Abcam (Cat. No. ab70161). For the secondary antibodies, anti-mouse-IgG-HRP and anti-rabbit-IgG-HRP were purchased from Cell Signaling (Beverly, MA, USA).
4
TRAIL-induced Apoptosis Regulation Mechanisms
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Cannabidiol and VAS2870 were purchased from Sigma (St. Louis, MO, USA). TRAIL and anti-DR5 were purchased from R&D Systems (Minneapolis, MN, USA). Anti-Bak, anti-Bcl-2, anti-Mcl-1, anti-Bcl-xL, and anti-DR4 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-XIAP, anti-NOXA, anti-BIM, anti-survivin, anti-Bid, anti-IRE1α, anti-phospho-IRE1α, anti-Bip, anti-GRP94, anti-ATF6, anti-eIF2α, anti-phospho-eIF2α, anti-CHOP, anti-cleaved PARP, anti-caspase-3, and anti-caspase-9 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The anti-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA). For the secondary antibodies, anti-mouse IgG horseradish peroxidase (HRP) and anti-rabbit IgG HRP were purchased from Cell Signaling Technology.
5
Spinal Cord Injury Protein Analysis
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Rats (n=5 in each group) were sacrificed 72 h after SCI, and protein homogenates were prepared from samples incubated in pyrolysis liquid for 2 h. The samples were centrifuged at 4°C and 12 000 rpm for 30 min, and the supernatant was collected and stored separately. The protein concentration of the sample supernatant was measured with a BCA kit (Beyotime Biotechnology). Total protein (60 μg) separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis was transferred to a polyvinylidene fluoride membrane (Beyotime Biotechnology). The membrane was blocked with 5% skim milk powder at room temperature for 2 h and incubated overnight with a primary antibody at 4°C. The primary antibodies included anti-P2X7 (1: 2000, Thermo Fisher Scientific), anti-NLRP1 (1: 1000, Abcam), anti-NLRP3 (1: 2000, Abcam), anti-XIAP (1: 1000, Cell Signaling Technology), anti-ASC (1: 2000, Novus Biologicals), anti-caspase-11 (1: 2000, Cell Signaling Technology), anti-caspase-1 (1: 2000, Abcam), and anti-β-actin (1: 5000, Proteintech Group) antibodies. Then, the membranes were washed with Tris-buffered saline containing Tween and incubated with secondary antibodies (1: 5000, Abcam) at room temperature for 2 h. Subsequently, protein bands were visualized with a chemiluminescence kit (Beyotime Biotechnology, Beijing), and images were collected using sensitive film and chemical imaging software.
6
Investigating Cell Death Pathways
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Cells were treated with recombinant human TNFα (R&D system), zyloxycarbonyl- Val-Ala-Asp(O-Me) fluoromethylketone (zVAD.fmk (abbreviated to Z) (Bachem, Bubendorf, Switzerland; no. N-1510), Necrostatin-1 (Nec-1) (Calbiochem, San Diego, CA, USA; no. 480065), BV6 (B) (Invitrogen), etoposide (ETPO), bortezomib (BOR) and dexamethasone (DEXA). The following antibodies were used for Western blotting: primary antibodies diluted 1:10,000 consist of (Goat anti-mouse; Jackson Immuno Research) and (Goat anti-rabbit; Jackson Immuno Research). Mouse anti FADD (Enzo Life Sciences no. Q13158, 1:1000), rabbit anti-RIP1 antibody (Cell Signaling Technology, 1:1000, no.610459), rabbit anti caspase 3 (Cell Signaling Technology, 1:1000, no.9662) and rabbit polyclonal anti-XIAP (Cell Signaling, 2042). Human SH-SY5Y neuroblastoma cells (ATCC, Manassas, VA, USA, RRID: CVCL_0019) were cultured in DMEM (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin, streptomycin (Gibco, Waltham, MA, USA) at 37 °C in a 5% CO2 incubator.
7
Protein Expression Analysis in Cell Lysates
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Cells were lysed by ice-cold cell lysis buffer (Cat# 17081, Intron Biotechnology, Seongnam, Korea) with the protease inhibitor cocktail. Protein concentrations were determined with a BCA assay kit (Cat# 23117, Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. Protein was separated via 10% SDS-PAGE, transferred to a PVDF membrane, and blocked with 5% non-fat milk. Membranes were incubated with anti-HOXB9 (Cat# PA5-101087, Thermo Fisher Scientific, Waltham, MA, USA), anti-ERCC-1 (Cat# ab129267), anti-MRP-2 (Cat# ab172630), anti-Bcl-2 (Cat# ab182858, abCAM, Cambridge, UK), anti-XIAP (Cat# 14334), anti-MMP-2 (Cat# 40994), anti-Bax (Cat# 5023), anti-cleaved caspase-3 (Cat# 9661), anti-cleaved PARP (Cat# 5625, Cell Signaling Technology, Inc., Danvers, MA, USA), and alpha-tubulin (Sigma-Aldrich, St. Louis, MO, USA) antibodies. Next, the membranes were incubated with HRP-conjugated anti-secondary Mouse IgG antibody (Cat# 7076) or HRP-conjugated anti-secondary Rabbit IgG antibody (Cat# 7074, Cell Signaling Technology, Inc., Danvers, MA, USA), and the visualized using Immobilin Forte Western HRP Substrate (Cat# WBLUF0100, Merk Millipore, Burlington, MA, USA).
8
Immunoblotting for Apoptosis Markers in AC16 Cells
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AC16 cells were homogenized in an ice-cold lysis buffer. After homogenization, the lysates were centrifuged. The supernatant was saved and separated by electrophoresis on SDS-PAGE and transferred onto polyvinylidene difluoride-plus membranes. After blocking buffer, the immunoblots were probed with anti-total caspase 3 (Cell Signaling) (lot: GR3356520-3), anti-XIAP (lot: GR3298310-4), and anti-actin (lot: GR333517-1) antibodies overnight at 4°C, followed by incubation with fluorescent-conjugated secondary antibodies at room temperature for 1 hour.
9
Modulation of Tongue Cancer Cell Apoptosis
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Tongue cancer cells were pretreated with or without an ERK inhibitor (U0126, Cell Signaling Technology, Danvers, MA, USA), a JNK1/2 inhibitor (JNK-IN-8, Calbiochem, San Diego, CA, USA), or a p38 inhibitor (SB203580, Calbiochem, San Diego, CA, USA) for 2 h and maintained in the presence or absence of DSK for 24 h. Total protein lysates (20 μg) were harvested and subjected to SDS-PAGE analyses [43 (link),44 (link)]. Specific antibodies targeting the following molecules were used for detection: Anti-cleaved Caspase-8 (#9496), Anti-cleaved Caspase-9 (#9505), Anti-cleaved Caspase-3 (#9664), Anti-Caspase-8 (#9746), Anti-Caspase-9 (#9502), Anti-PARP (#9542), Anti-Phospho-Erk1/2 (#4370), Anti-Erk1/2 (#9102), Anti-Phospho-JNK (#4668), Anti-JNK2 (#9258), Anti-c-IAP1 (#7065), and Anti-XIAP (#2045) antibodies from Cell Signaling Technology (Danvers, MA, USA); Anti-Caspase-3 (610323), Anti-phospho-p38 (612281), and Anti-p38 (612168) antibodies from BD biosciences (San Jose, CA, USA); Anti-β-actin (ab8226) and anti-HO-1 (ab68477) antibodies from Abcam (Cambridge, UK); Anti-Nrf2 (GTX55732) antibodies from GeneTex (Irvine, CA, USA); and HRP-conjugated secondary antibodies (Dako Corporation, Carpinteria, CA, USA). Densitometry data of immunoblots were generated and analyzed by ImageJ software.
10
Protein Expression Analysis in Cell Lysates
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Total cell lysate was prepared using lysis buffer containing 10 µg/ml phenylmethylsulfonyl fluoride (PMSF), 20 µg/ml protease inhibitor cocktail (PIC), 20 µg/ml sodium fluoride and 10 µg/ml sodium orthovanadate (Na3VO4). The protein concentration in the cell lysate was estimated using Lowry protein assay and 25 µg of protein was aliquoted to run a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a 4–20% Tri-glycine gel. Data from the gel were transferred onto a nitrocellulose membrane and incubated with the anti-cyclin D1 (sc-450), anti-Cdk2 (sc-6248), anti-Cdk4 (sc-260), anti-p27 (sc-393380), anti-GAPDH (sc-166545), anti-Bax (sc-493), anti-Bcl2 (sc-7382), anti-c-Myc (sc-40), anti-cyclin E (sc-248) were procured from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Additionally anti-XIAP (#2042), anti-FOXO3a (#12829), anti-cleaved caspase-3 (#9661), anti-cleaved PARP (#5625) procured from Cell Signaling Technology (Danvers, MA, USA) and anti-β-actin (#A1978) were procured from Sigma-Aldrich. Secondary antibodies used were anti-mouse (#HAF007) anti-rabbit (#HAF008) from R&D Systems (Minneapolis, MN, USA). All antibodies were diluted to 1:1,000 dilution of stock solution of 1 mg/ml. Densitometric analysis of the protein bands were performed using the Kodak 2000R imaging system digitalized scientific software program.
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