BCL1 cells, grown in culture plates, were treated with or without Pt(S-pr-thiosal)2 (0.05 mg/mL) for 12 h. The treated cells were fixed and permeabilized with permeabilization buffer (BD Bioscience) and incubated with antibodies specific for Bcl-2 (11-6992-42, Thermo Fisher Scientific, Cambridge, UK), Noxa (ab13654, Abcam, Cambridge, UK), STAT3 (IC1799G, Novus Biologicals, San Diego, CA, USA), p16 (ab211542, Abcam, Cambridge, UK), p21 (ab188224, Abcam) and p27 (ab215434, Abcam), cyclin D3 (ab28283, Abcam Cambridge, UK), cyclin E (MA5-14336, Thermo fisher scientifics, Waltham, MA, USA), Ki-67 (151212, BioLegend, San Diego, CA, USA), Noxa (ab13654, Abcam) and Mcl-1 (ab32087, Abcam). For staining p16, p21, and p27 cells were additionally incubated with secondary goat anti-mouse IgG FITC (ab6785, Abcam) or donkey anti-rabbit IgG (ab150073, Abcam). Flow cytometry was performed on FACSCalibur flow cytometer (BD Biosciences). The data were analyzed using FlowJo (Tree Star).
Ab13654
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab13654 is a primary antibody used for immunological detection. It is a purified monoclonal antibody produced in mouse.
Automatically generated - may contain errors
Lab products found in correlation
Ab32087, by Abcam (2 mentions)
Ab182858, by Abcam (2 mentions)
Permeabilization buffer, by BD (1 mentions)
Ic1799g, by Novus Biologicals (1 mentions)
Ma5 14336, by Thermo Fisher Scientific (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Ab150073, by Abcam (1 mentions)
Ab6785, by Abcam (1 mentions)
Ab188224, by Abcam (1 mentions)
Ab211542, by Abcam (1 mentions)
Ab28283, by Abcam (1 mentions)
Ab215434, by Abcam (1 mentions)
Ab32539, by Abcam (1 mentions)
Ab25901, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Lehd para nitroaniline lehd p na, by Thermo Fisher Scientific (1 mentions)
Ietd para nitroaniline ietd p na, by Thermo Fisher Scientific (1 mentions)
Devd p na, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Dmem ham s f12, by Thermo Fisher Scientific (1 mentions)
6 protocols using ab13654
1
Investigating Apoptosis Pathway in BCL1 Cells
2
Apoptosis Pathway Analysis Protocol
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Dulbecco’s Modified Eagle Medium—high glucose (DMEM-HG) (12800-58), Roswell Park Memorial Institute (RPMI)-1640, DMEM/Ham’s F-12 (GIBCO-Invitrogen, Carlsbad, CA, USA), fetal bovine serum (FBS), phosphate-buffered saline (PBS)and trypsin-EDTA solution were purchased from Gibco (Grand Island, NY, USA) Dimethyl sulfoxide (DMSO), and sulforhodamine B (SRB) was purchased from Sigma Chemical, Inc. (St Louis, MO, USA) The substrate of caspase-9 (LEHD-para-nitroaniline; LEHD-p-NA), caspase-8 (IETD-para-nitroaniline; IETD-p-NA), caspase-3 (DEVD-para-nitroaniline; DEVD-p-NA), and SuperSignal West Pico Chemiluminescent Substrate were obtained from Invitrogen (Thermo Fisher Scientific Inc., Waltham, MA, USA). Primary antibodies against caspase-9 (ab32539), caspase-8 (ab25901), caspase-7 (ab25900), BAX (ab32503), Bcl-xl (ab32370), Bid (ab2388), Noxa (ab13654), actin (ab8227) and peroxidase-labeled secondary antibodies; anti-rabbit IgG (ab97051), anti-mouse IgG (ab97046) were purchased from Abcam (Cambridge, UK). Protease inhibitor was obtained from Roche Diagnostics, Mannheim, Germany.
3
Cell Cycle Progression and Apoptosis Analysis
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Cells were incubated for at least 24 h prior to irradiation and/or PM2 treatment. Twenty micrometer PM2 was added with a 3-h delay after irradiation. Cells were collected and fixed in 70% ethanol at 6, 12, 24, 48, 72, and 96 h post-irradiation and stored at −20°C. For cell cycle analyses, 6, 12, 24, and 48 h samples were rehydrated and washed twice with PBS and stained with DAPI (Sigma Aldrich Sweden, Stockholm, Sweden) using 1 μg/ml for 30 min. 24, 48, 72, and 96 h samples were stained with caspase-3 and Noxa (ab13847 (1:500) and ab13654 (1:1,000), AbCam, Cambridge, UK) overnight at 4°C, followed by incubation with fluorescent labeled secondary antibodies for 90 min (ab 1:400) at room temperature. Analyses were performed using a BD LSR Fortessa flow cytometer (Becton Dickinson Biosciences, San Jose, USA). Data analyses for cleaved caspase-3 and Noxa were performed with BD FACSDiVa (Becton Dickinson Biosciences, San Jose, USA), while cell cycle analyses of exclusively viable cells were performed using FlowJo (Becton, Dickinson Biosciences, San Jose, USA). Coefficient of variation (CV) values, were below seven for all samples.
4
Protein Expression Analysis of Intestinal Tissues
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Total protein was extracted from intestinal tissues or IEC-6 cells using RIPA lysis buffer (Beyotime, Shanghai, China) and 1% PMSF (Beyotime, Shanghai, China). Denatured protein samples were separated with 10% SDS-PAGE and transferred to 0.45 μm polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were incubated with primary antibodies overnight at 4 °C. After washing, the membranes were incubated with a 1:5000 dilution of HRP conjugated secondary antibodies. The following primary antibodies were used: anti-VDR (1:2000, 12550S, Cell Signaling Technology, Danvers, MA, USA), anti-Pmaip1 (1:500, ab13654, Abcam, Cambridge, MA, USA), anti-Cleaved Caspase-3 (1:2000, 9664S, Cell Signaling Technology, Danvers, MA, USA) and anti-β-Actin (1:1000, 4970S, Cell Signaling Technology, Danvers, MA, USA). The Western blot bands were quantified using Image J software, and the quantitative results were expressed by integral optical density (IOD).
5
Western Blot Analysis of Apoptosis Markers
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Western blot analysis was performed as described previously [42 (link)]. Antibodies against full-length and cleaved caspase-9 (PA5-17913, Invitrogen, Waltham, Massachusetts, USA), full-length and cleaved caspase-3 (9662, Cell Signaling Technology, Danvers, MA, USA), full-length and cleaved PARP (9542, Cell Signaling Technology, Danvers, MA, USA), Mcl-1 (4572, Cell Signaling Technology, Danvers, MA, USA), Bcl-2 (ab182858, Abcam, Cambridge, UK), Bcl-XL (2762S, Cell Signaling Technology, Danvers, MA, USA), p53 (sc-126, Santa Cruz Biotechnology, Dallas, Texas, USA ) and NOXA (ab13654, Abcam, Cambridge, UK) were diluted 1:1000. Secondary antibodies against rabbit IgG (A0545, Sigma-Aldrich, St. Louis, MI, USA) were diluted 1:10,000 and secondary antibodies against mouse IgG (A9044, Sigma Aldrich, St. Louis, MI, USA) were diluted 1:10,000.
6
Immunohistochemistry for Apoptosis Regulators
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Immunohistochemistry (IHC) was carried out as followed. In brief, rst, the para n-embeded samples were sectioned into 4µm-thick sections and dewaxed in xylene, rehydrated in rinsed graded ethanol solutions. Endogenous peroxidase activity was then blocked with 3% hydrogen peroxide solution for 10 minutes, rinsed in phosphate buffered saline (PBS) 3 times for 5 min each. Then the tissue sections were heated at 100℃ for 5 minutes in citrate (10mmol/L, pH 6.0) solution to retrieve the antigens. After cooling to room temperature, serum blocker was added to block non-speci c antigen, and were incubated with the primary antibody Bcl-2 (ab182858; Abcam, Cambridge, MA, USA, 1:1000 dilution), Bcl-xL (cat#2764, Cell Signaling Technology, USA, 1:3000 dilution), Mcl-1(ab32087; Abcam, Cambridge, MA, USA, 1:200 dilution), Noxa (ab13654; Abcam, Cambridge, MA, USA, 1:2000 dilution), PUMA (ab33906; Abcam, Cambridge, MA, USA, 1:200 dilution) at 4℃ overnight, followed by washing with PBS for three times, biotinylated goat anti-mouse IgG (1:400; Sigma, St. Louis, MO, USA) or goat anti-rabbit IgG (1:400; Sigma) for 30 min at room temperature. Finally, the signal was developed for visualization with 3,3'-diaminobenzidine tetrahydrochloride (DAB, DAKO, GLOSTRUP, Denmark), and all of the slides were counterstained with hematoxylin.
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