The following antibodies were used: anti-DR4 (307208 for flow cytometry; BioLegend, San Diego,CA; ab8414 for western blotting; Abcam, Cambridge, UK), anti-DR5 (119906 for flow cytometry; BioLegend; ab8416 for western blotting, Abcam), anti-DcR1 (ab2087; Abcam), anti-DcR2 (ab2019; Abcam), anti-CXADR (05-644; Millipore, Billerica, MA), anti-Ad5 (ab6982; Abcam), anti-caspase-3 (sc-56055; Santa Cruz Biotechnology; Santa Cruz, TX), anti-c-caspase-3 (610322; Becton Dickinson, Franklin Lakes, NJ), anti-PARP (100984-T46; Sino Biological, Beijing, China), anti-FLIP (ab56531; Abcam), anti-tubulin (ab15246; Abcam), anti-CD45 (10086-MM05-F; Sino Biological), and anti-CD33 (12238-MM09; Sino Biological). Rh2 (MB6870) was purchased from Meilunbio (Dalian, China).
Ab8416
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab8416 is a monoclonal antibody that targets the Phosphorylated Histone H3 (Ser10) protein. It is designed for use in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab8414, by Abcam (3 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Ab6982, by Abcam (1 mentions)
Ab15246, by Abcam (1 mentions)
Ab16066, by Abcam (1 mentions)
Ab172476, by Abcam (1 mentions)
Ab180710, by Abcam (1 mentions)
Ab15580, by Abcam (1 mentions)
Sc 32233, by Santa Cruz Biotechnology (1 mentions)
Poly adenosine diphosphate ribose, by Cell Signaling Technology (1 mentions)
Bcl 2 sc 509, by Santa Cruz Biotechnology (1 mentions)
Prestoblue cell viability reagent, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Methanol, by Avantor (1 mentions)
Ab150113, by Abcam (1 mentions)
Ab5831, by Abcam (1 mentions)
Ab1431, by Abcam (1 mentions)
8 protocols using ab8416
1
Antibody Analysis of TRAIL Receptors
2
Lentiviral Protein Expression and Analysis
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The cells were lysed with RIPA buffer for 30 min at 4°C for protein extraction after infection with lentivirus. A BCA assay was applied to determine the protein concentrations. The same amounts of protein were separated on 12.5% SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with anti-CCND1 (#2978) or anti-CDH1 (#14472) primary antibodies (Cell Signaling Technologies (CST), USA) as well as other antibodies, including those against MKI67 (ab15580), TNFRSF10B (ab8416), FOXM1 (ab180710), RRM2 (ab172476), HIF1A (ab16066) (Abcam, UK), and GAPDH (SC-32233) (Santa Cruz Biotechnology, USA). Anti-CHPF antibody (Orb127868) was purchased from Biorbyt Ltd. (UK). The membranes were then incubated with HRP-conjugated antibodies (CST, #7076, #7074).
3
Apoptosis-Inducing Mechanism Investigation
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Prestoblue Cell Viability reagent, RPMI-1,640 and fetal bovine serum (FBS) were purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and methanol was purchased from Avantor Performance Materials (Center Valley, PA, USA). Primary antibodies against caspase-3 (no. 9662), caspase-8 (no. 4927) and caspase-9 (no. 9508), poly adenosine diphosphate ribose polymerase (PARP; no. 9532), B-cell lymphoma-extra large (Bcl-xL; no. 2764), survivin (no. 2808), Bak (no. 12105), Bid (no. 2003), death receptor 4 (DR4; no. 42533) and β-actin (no. 3700) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibodies against B-cell lymphoma 2 (Bcl-2; sc-509) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Antibodies against death receptor 5 (DR5; ab8416) were purchased from Abcam (Cambridge, MA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin (Ig)G (AP124P) and goat anti-rabbit IgG (AP132P) secondary antibodies were obtained from EMD Millipore (Billerica, MA, USA).
4
Quantifying TRAIL Death Receptor (DR5) Expression
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The TRAIL death receptor (DR5) was analyzed by immunofluorescence. Cell lines were cultured in Lab-Tek® microchambers (Nunc®, Thermo Scientific, Waltham, Massachusetts, USA) and, after 24 h, were fixed with methanol-acetone (1:1 v/v) and washed three times with Tris-Buffered saline Tween 20 (TBST). Slides were then incubated with primary antibodies anti-DR5 (ab8416, Abcam, Burlingame, CA, USA) (1:1000) and incubated at 4 °C for 12 h. The slides were washed again three times with TBST. The secondary antibody Alexa Fluor® 488 (ab150113, Abcam, Burlingame, CA, USA) (1:1000) was added and incubated for 1 h at 25 °C. All antibodies were diluted in phosphate saline buffer (PBS). VECTASHIELD® 25 µL mixed with 4′,6′-diamino-2-phenilindol (DAPI) (Vector Laboratories, Inc. 6737 Mowry Ave Newark, CA 94560 USA) was added as a mounting medium. Slides were observed with a ZEISS Imager.A2 epifluorescence microscope (Carls ZEISS, Alemania). Five pictures were taken per slide with an AxioCam MRc coupled to the microscope. Images were analyzed with ZEISS ZEN Blue and Image J software. The expression percentage was calculated with relative luminescence units as the (positive Red-Green/DAPI signal) × 100.
5
Protein Expression Analysis in Liver Tissues
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The liver tissues or cells were suspended and lysed in radio‐immunoprecipitation assay buffer (Beyotime) supplemented with protease inhibitor cocktail (Sigma, St. Louis, MO, USA). Protein extractions were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Millipore). The membrane was blocked with 5% (w/v) reagent‐grade non‐fat milk (Cell Signaling Technology, Santa Cruz, CA, USA) and incubated with primary antibody against α‐SMA (ab5831; Abcam), p53 (ab1431; Abcam), caspase 3 (ab13847; Abcam), Bid (ab10640; Abcam), DR4 (ab209412; Abcam), DR5 (ab8416; Abcam), uPA (sc‐59727; Santa Cruz, USA), PAI‐1 (sc‐5297; Santa Cruz, USA), β‐crystallin (ab13496; Abcam), collagen I (ab34710; Abcam), collagen III (ab7778; Abcam) or glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH, ab8245; Abcam) at 4°C overnight followed by secondary antibody incubation. The protein bands were visualized using ClarityTM Western ECL substrate (Bio‐Rad, Hercules, CA, USA). The protein level was quantified using Image J software normalized with GAPDH.
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
Western Blot Analysis of Apoptosis Regulators
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Total protein was extracted using RIPA buffer containing proteases and phosphatase inhibitors (Thermo Fisher Scientific, Waltham, MA, USA), and protein concentration was determined with the Bradford protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Proteins were separated by electrophoresis on a 10–13% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (Amersham International, Little Chalfont, UK). Membranes were blocked with 5% bovine serum albumin (BSA; bioWORLD, Dublin, OH, USA) in Tris-buffered saline with tween® 20 (TBST) for 1 h at room temperature. Membranes were subsequently washed with Tris-buffered saline with tween® 20 detergent (TBST) and incubated with primary antibodies to tristetraprolin (ab33058; Abcam, Cambridge, UK), DR4 (ab8414; Abcam), DR5 (ab8416; Abcam), and β-actin (sc-47778; Sigma-Aldrich, St. Louis, MO, USA), and diluted in 5% BSA/TBST overnight at 4 °C. Membranes were washed with TBST. The secondary antibody (anti-mouse or anti-rabbit IgG HRP conjugate; Bethyl Laboratories, Montgomery, TX, USA) was diluted 2000-fold in TBST and applied to cells for 1 h. After washing with TBST, specific binding of antibodies was detected using an ECL kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s protocol.
8
Hypoxia-Induced Protein Expression Analysis
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After hypoxia treatment, total protein was extracted using a Western & IP cell lysis kit (Beyotime)containing1mM PMSF. Nuclear and cytoplasmic proteins were separated using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime) according to the manufacturer's instructions.Whole cell extracts were then separated by 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis and electrotransferred to a polyvinylidenedi oride (PVDF) membrane (Beyotime). Afterblocking, the membranes were incubated at 4 o C overnight with rabbit polyclonal Antibodies against Vacuolar H + ATPase (VMA21/V-ATPase, 1:1000 dilution,ab242099, Abcam, Cambridge, UK), TUBB1/β-tubulin (1:1000 dilution, Sigma,T7816), laminB(1:1000 dilution, Sigma, SAB1306342), TFEB(1:1000 dilution, SAB2107961, Sigma), LAMP 2 (1:1000 dilution, AF1036, Beyotime), JNK1 (1:1000 dilution, 3708, CST), Phospho-JNK1 (1:1000 dilution, 9255, CST), c-Jun (1:1000 dilution, 9165, CST), Phospho-c-Jun(1:1000 dilution, 9261, CST), DR 5 (1:500 dilution, ab8416,Abcam, Cambridge, UK),Caspase-8 (1:1000 dilution, 4790, CST) and β-actin(1:1000 dilution, sc-8432, Santa Cruz), Then the membranes were washed with TBST and incubated with horseradish peroxidase-linked secondary antibodies (ZDR-5306, ZDR-5307,Zhong Shan, Beijing, China). After being washed with TBST, immunoreactive bands were visualized using NBT/BCIP (Beyotime)as substrate.
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