Cal-27 cells treated for 24h or 48h with vehicle, CIDD-24, CIDD-99, and CIDD-111 at the indicated concentrations were harvested and lysed in Laemmli Lysis Buffer. Cell lysates (40μg) were separated using electrophoresis in 10% SDS-PAGE, separated proteins were transferred to PVDF membrane, and membrane blocked in 5% milk solution. Rabbit polyclonal antibodies (Cell Signaling, Danvers, MA, USA) against cPARP (#5625S; 1:1250), Bip (#3177S; 1:1000), and CHOP (#2895T; 1:1000) and rabbit monoclonal anti-α/β-tubulin antibody (Cell Signaling, #CS2148; 1:1,000) were diluted in 6ml diluent (1% milk in PBS-0.1% Tw-20) and incubated overnight at 4°C (12 (link)). Membranes were washed with PBS-Tw-20, incubated with ECL Plus detection solution (GE Healthcare, South San Francisco, CA, USA) for 1 min, and signal was detected by radiographic exposure for 30 seconds.
Rabbit polyclonal antibodies
Manufactured by Cell Signaling Technology
Sourced in United States
Rabbit polyclonal antibodies are a type of immunoglobulin produced by rabbits in response to a specific antigen. These antibodies are a heterogeneous mixture of immunoglobulins that recognize multiple epitopes on the target antigen.
Automatically generated - may contain errors
Lab products found in correlation
Tb green premix ex taq 2 tli rnaseh plus, by Takara Bio (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Rabbit monoclonal anti α β tubulin antibody, by Cell Signaling Technology (1 mentions)
Ecl plus detection solution, by GE Healthcare (1 mentions)
Odyssey clx, by LI COR (1 mentions)
Nucleoprotein extraction kit, by Sangon (1 mentions)
Alexa fluor 594 goat anti mouse antibody, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence substrate, by Cytiva (1 mentions)
Aqueous methylcellulose, by Merck Group (1 mentions)
Activated charcoal, by Merck Group (1 mentions)
Primescript 1st strand cdna synthesis kit, by Takara Bio (1 mentions)
Polyvinylidene difluoride pvdf membrane, by Cytiva (1 mentions)
Luciferase assay kit, by Promega (1 mentions)
β actin primary antibody, by Santa Cruz Biotechnology (1 mentions)
D luciferin potassium salt, by GoldBio (1 mentions)
Psv β galactosidase vector, by Promega (1 mentions)
P ikkα β, by Santa Cruz Biotechnology (1 mentions)
Ikkα β, by Santa Cruz Biotechnology (1 mentions)
11 protocols using rabbit polyclonal antibodies
1
Quantification of Apoptosis Markers
2
Western Blot Analysis of Signaling Proteins
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The liver proteins were extracted by using the tissue protein extraction solution or the nucleoprotein extraction kit (Sangon Biotech, Shanghai, China). The protein content was measured as previously described. The same amount of total protein from each sample was electrophoresed into SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were probed using rabbit polyclonal antibodies (Cell Signaling) against p-ERK1/2 (Thr202/Tyr204) (#9101), ERK1/2 (#9102), p-JNK(Thr183/Tyr185)(#9251), JNK (#9252), p-AKT (Ser473) (#9271), AKT(#4691), p-NF-κB (Ser468) (#3039), NF-κB (#3034) and p-AMPK (Thr172) (#2535), AMPK (#2532) overnight at 4 °C. After washing five times with TBST, the membranes were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) for 1 h. The images of specific proteins were detected and analyzed using the LI-COR Odyssey CLx.
3
Antibody Panel for Protein Analysis
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Mouse monoclonal antibodies for PFKM (sc-67028, 1:1,000 for immunoblotting), β-catenin (E-5, sc-7963, 1:200 for immunoblotting and 1:50 for immunofluorescence), and c-Myc (9E10, sc-40, 1:200 for immunoblotting) and polyclonal antibody for cyclin D1 (H-295, sc-753, 1:200 for immunoblotting) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal antibodies recognizing PFKP (12746, 1:1,000 for immunoblotting), PFKL (8175, 1:1,000 for immunoblotting), and β-catenin pS552 (9566, 1:1,000 for immunoblotting) were purchased from Cell Signaling Technology (Danvers, MA). Mouse monoclonal antibody for tubulin (clone B-5-1-2, T6074, 1:5,000 for immunoblotting) was purchased from Sigma (St. Louis, MO). Mouse monoclonal antibody for PCNA (610665, 1:1,000 for immunoblotting was purchased from BD Biosciences (San Jose, CA). Human recombinant EGF (01-407) was obtained from EMD Millipore (Billerica, MA). Hygromycin (400053), puromycin (540222), and G418 (345810) were purchased from EMD Biosciences (San Diego, CA). HyFect transfection reagents (E2650) were obtained from Denville Scientific (Metuchen, NJ). DAPI and Alexa Fluor 594 goat anti-mouse antibody were purchased from Molecular Probes (Eugene, OR).
4
Signaling Pathways in Intestinal Inflammation
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The chemicals were obtained from the following sources: Loperamide hydrochloride (Lop), prucalopride, sodium chloride, activated charcoal, and aqueous methylcellulose from Sigma-Aldrich (St Louis, MO, USA). Rabbit monoclonal antibody against phospho-extracellular signal-regulated kinase (ERK) 1/2 (Thr202/Tyr204), rabbit polyclonal antibodies against phospho-p38 (Thr180/Tyr182) and phospho-SAPK/JNK (Thr183/Tyr185), horseradish peroxidase (HRP)-conjugated anti-rabbit, and anti-mouse immunoglobulin G (IgG) antibodies were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). Rabbit anti-c-Kit and anti-SCF antibodies were obtained from Abcam (Cambridge, MA, USA). The BCA protein assay kit was obtained from Thermo Scientific (Waltham, MA, USA). The polyvinylidene difluoride (PVDF) membrane and enhanced chemiluminescence substrate were obtained from Amersham Pharmacia Biotech. The Hybrid-R™ RNA extraction kit was purchased from GeneAll Biotechnology Co., Ltd (Seoul, Korea). The PrimeScript™ 1st strand cDNA Synthesis kit and TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) were acquired from Takara (Takara, Japan). All other chemicals were of analytical grade or complied with the standards.
5
Signaling Pathways Modulation by CCN6
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We purchased p85α, Akt1, p-IKKα/β (Ser180/Ser181), p-IκBα, p65 (Ser536), IKKα/β, IκBα, p65, CCN6, and β-actin primary antibodies (Santa Cruz Biotechnology, CA, USA), and rabbit polyclonal antibodies specific for p-p85 (Y458), p-Akt (S473), p-mTOR (Ser2448), and mTOR (Cell Signaling Technology, Danvers, MA, USA). Recombinant human CCN6 was purchased from PerpoTech (Rocky Hill, NJ, USA) and CCN6 short hairpin (sh)RNA expression plasmids from RNAiCore (Taipei, Taiwan). The D-Luciferin potassium salt was purchased from Gold Biotechnology (St. Louis, MO, USA). Lipofectamine 2000 and TRIzol were purchased from Life Technologies (Carlsbad, CA, USA). Dharmacon Research (Lafayette, CO, USA) supplied ON-TARGETplus siRNAs. Gibco-BRL life technologies (Grand Island, NY, USA) supplied DMEM, α-MEM, fetal bovine serum (FBS), and all other cell culture reagents. The Matrigel was purchased from BD (Biosciences, Bedford, MA, USA) and Promega (Madison, WI,) supplied the pSV-β-Galactosidase Vector and luciferase assay kits. All other USA chemicals or inhibitors that we used were supplied by Sigma-Aldrich (St. Louis, MO, USA).
6
Antibody and Reagent Procurement Protocol
7
Antibody Characterization for Signaling Pathways
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Rabbit polyclonal antibodies that recognize β‐actin, phospho‐(Ser536) and total NF‐κB; phospho‐(Thr‐180/Tyr‐182) and total p38 mitogen‐activated protein kinase; and phospho‐(Thr‐202/Try‐204) and total extracellular signal–related kinase 1/2 were purchased from Cell Signaling Technology. Horseradish peroxidase–conjugated secondary antibodies that recognize rabbit IgG were purchased from KLP Antibodies. Antibodies recognizing β‐tubulin (rabbit) and lamin A/C were purchased from Santa Cruz Biotechnology.
8
Aβ(1-42) Induced Src Phosphorylation
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Cells were incubated with 10 μM Aβ(1-42) for 30 min and then lysed in the RIPA buffer (25 mM tris-HCl, pH 7.6, 150 mМ NaCl, 1% Nonidet-P40, 0.1% SDS, 1% sodium deoxycholate) containing 1 μM of PMSF with stirring at 4 °C for 1 h. The probes were then centrifuged at 13000 g for 10 min and the supernatant was collected. Proteins of cell lysates were separated on SDS-PAGE and transferred to a PVDF membrane. After membrane blocking in 5% nonfat milk in PBST, the detection of phospho (Tyr 416) Src and total Src was carried out by incubating the membrane in the solution of appropriate rabbit polyclonal antibodies (both from Cell Signaling Technology) in PBST. The level of β-actin was also estimated using mouse monoclonal anti-β-actin antibody (Ambion). Visualization of the proteins was performed by the appropriate horseradish peroxidase-conjugated secondary antibodies provided by the enhanced chemiluminescence SuperSignal ™ West Femto Maximum Sensitivity Substrate kit (ThermoScientific). Chemiluminescence was detected using Bio-Rad ChemiDoc MP instrument. Densitometric analysis was performed with Image Lab program (Bio-Rad) and the results were expressed as ratio of phospho-Src to total Src band intensity (phospho-Src/Src).
9
Atractylenolide I Cytotoxicity Assay
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RPMI-1640 medium, fetal bovine serum, penicillin, streptomycin and trypsin were purchased from Gibco BRL (Grand Island, NY, USA). Atractylenolide I, MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] were purchased from Sigma (St. Louis, MO, USA). De-ionized water (ddH2O) was collected from Millipore water purification system (Millipore, Milford, MA, USA). All rabbit polyclonal antibodies were purchased from Cell Signaling Technology (Dancers, MA, USA), except anti-caspase 8 antibody, which was purchased from Santa Cruz (Dallas, TX, USA).
10
Osteoclast Differentiation and Signaling
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Recombinant soluble RANKL ligand (sRANKL) was acquired from PeproTech EC Ltd. (London, UK). An Acid Phosphatase Assay Kit, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), leukocyte acid phosphatase, 4-6-diamidino-2-phenylindole (DAPI), ascorbic acid, and β-glycerophosphate were purchased from Sigma-Aldrich Fine Chemicals (Saint Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), minimum essential medium alpha (α-MEM), and fetal bovine serum (FBS) were purchased from Welgene Bioscience (Daegu, Korea). TRIZOL reagent was purchased from JBI (Seoul, Korea) and TOPscript™ RT DryMIX (dT18 plus) was purchased from Enzynomics (Daejeon, Korea). TB Green® Pre-mix Ex Taq™ II (Tli RNaseH Plus) was purchased from Takara (Tokyo, Japan). Primary antibodies for phospho-JNK, JNK, phospho-p38, p38, phospho-ERK, ERK, BIP, p-eIF2α, eIF2α, and rabbit polyclonal antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Catalase (CAT) and superoxide dismutase (SOD) antibodies were acquired from BD biosciences (San Jose, CA, USA). An anti-nuclear factor of activated T cells-c1 (NFATc1) antibody was acquired from BD biosciences (San Jose, CA, USA). The enhanced chemiluminescence (ECL) Western blotting detection system was purchased from Advansta Inc. (San Jose, CA, USA).
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