The following reagents and antibodies were used in this study: salidroside (C14H20O7, CAS#:10338–51–9, Nanjing, China); recombinant human TNF-α, type II collagenase, and Safranin-O/Fast Green (Sigma-Aldrich, St. Louis, MO, USA).primary antibody against p65(Cat# 59674, RRID:AB_2799570), COX-2(Cat# 12282, RRID:AB_2571729), IκBα(Cat# 4812, RRID:AB_10694416), and XBP-1s (Cat# 12782, RRID:AB_2687943) were purchased from Cell Signaling Technology(Danvers, MA, USA); anti-iNOS (Cat# SAB4502012, RRID:AB_10744871) were bought from Sigma-Aldrich; antibodies against PCAF(Cat# ab110421, RRID:AB_11156343), H3K9ac(Cat# ab4441, RRID:AB_2118292), lamin-B1(Cat# ab22137, RRID:AB_446813), GAPDH(Cat# ab9485, RRID:AB_307275), cleaved caspase 3(Cat# ab2302, RRID:AB_302962), cleaved caspase 12(Cat# ab62484, RRID:AB_955729), cytochrome C(Cat# ab133504, RRID:AB_2802115), and cleaved PARP (Cat# ab32064, RRID:AB_777102) were obtained from Abcam (Cambridge, UK). Alexa Fluor 594- and Alexa Fluor 488-labelled anti-rabbit goat immunoglobulin G (H + L) inferior antibody were bought from Jackson Immuno Research (West Grove, PA, USA). Gibco (Carlsbad, CA, USA) provided cell-culture reagents.
Cleaved caspase 12
Manufactured by Abcam
Sourced in United Kingdom, Italy, United States
Cleaved caspase 12 is a protein involved in the apoptosis (programmed cell death) pathway. It is a marker of endoplasmic reticulum (ER) stress-induced apoptosis.
Automatically generated - may contain errors
Lab products found in correlation
Grp78, by Abcam (3 mentions)
Cleaved caspase 3, by Abcam (2 mentions)
Anti inos, by Merck Group (1 mentions)
Gapdh, by Abcam (1 mentions)
Cox 2, by Cell Signaling Technology (1 mentions)
Safranin o fast green, by Merck Group (1 mentions)
Cytochrome c, by Abcam (1 mentions)
Lamin b1, by Abcam (1 mentions)
Primary antibody against p65, by Cell Signaling Technology (1 mentions)
Recombinant human tnf α, by Merck Group (1 mentions)
Collagenase type 2, by Merck Group (1 mentions)
Cleaved parp, by Abcam (1 mentions)
Xbp1s, by Cell Signaling Technology (1 mentions)
H3k9ac, by Abcam (1 mentions)
Hybond p pvdf membrane, by GE Healthcare (1 mentions)
Hyperfilm mp, by GE Healthcare (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Criterion tris hcl precast gel, by Bio-Rad (1 mentions)
Peroxidase conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
7 protocols using cleaved caspase 12
1
Chondroprotective Effects of Salidroside
2
Quantitative Western Blot Analysis
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Cells were treated with CP and, after different times, were collected, centrifuged and washed with ice cold phosphate-buffered saline (PBS). The pellet was then resuspended in lysis buffer as described [47 ]. The protein concentration in the supernatant was determined using the BCA protein assay (Pierce, Italy). Equal amounts of protein (10 μg) were resolved using SDS-PAGE gel electrophoresis (Criterion precast Tris-HCl gel, BioRad, Italy) and transferred to PVDF Hybond-p membranes (GE Healthcare, Italy). Membranes were blocked with 2% ECL-Blocking Solution (GE Healthcare, Italy) for 2 hours at room temperature. Membranes were then incubated with primary antibodies against Bcl-2, PARP, procaspase-9, cleaved caspase-7, GRP78, (Cell Signaling, Italy), cleaved caspase-12 (Abcam, UK), β-actin (Sigma Aldrich, Italy), and cleaved caspase-3 (Novus Biologicals, Italy) overnight. Membranes were then incubated with peroxidase-conjugated secondary antibodies (Invitrogen, Italy) for 60 min. All membranes were visualized using ECL Select (GE Healthcare, Italy) and exposed to Hyperfilm MP (GE Healthcare, Italy). To ensure equal protein loading, each membrane was stripped and reprobed with anti-β-actin antibody.
3
Sciatic Nerve Injury Protein Analysis
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The contusion epicenter of sciatic nerve tissue (2 cm) was lysed in RIPA buffer containing phenylmethanesulfonyl fluoride (the volume ratio is 100:1) for 30 min and clarified by centrifugation at 12,000 rpm for 15 min. Then, the extracts were quantified with Carmassi Bradford reagents (Thermo, Rockford, IL, USA). 80 μg protein was separated using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto PVDF membranes (Millipore, USA). After blocking with 5% skim milk for 1.5 h, membranes were probed with primary antibody (GRP-78, ATF-6 and Cleaved-caspase 12, 1:1000, Abcam; XBP-1 and CHOP 1:300, Santa Cruz; GAPDH, 1:10000, Bioworld). Subsequently, blots were incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies (1:10000) [50 (link), 51 (link)]. Finally, the immunoreactive bands were scanned via optical density measurements using the ChemiDoc XRS+ Imaging System (Bio-Rad). Each sample was blotted 3 independent times, and the densitometric values of the bands were quantified with the Image Lab software (Bio-Rad).
4
Endoplasmic Reticulum Stress Response Mechanisms
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All reagents used in this study were commercially available. aFGF was purchased from Key Laboratory of Biotechnology and Pharmaceutical Engineering, Zhejiang, China, and the In Situ Cell Death Detection Kit was purchased from Roche (South San Francisco, CA, USA). Foetal bovine serum and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Invitrogen (Carlsbad, CA, USA). Antibodies against ATF-6, ATF-4, GRP-78, PDI, XBP-1 and cleaved caspase 12 were purchased from Abcam (Cambridge, MA, USA). Anti-β-actin, CHOP, microtubule-associated protein 2 (MAP-2) and myelin basic protein (MBP) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-cleaved caspase 3 antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). The appropriate secondary antibodies were purchased from Abcam or Santa Cruz Biotechnology.
5
NBP Modulates HBMEC Barrier Function
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NBP, a yellow oil-like liquid with a purity of 98.6%, was got from (Shijiazhuang Pharmaceutical Group Enbipu Pharmaceutical Co., Ltd, Shijiazhuang, Hebei, China). Human brain microvascular endothelial cell (HBMEC) was bought from ScienCell Research Laboratories (San Diego, CA, USA). Texas red-conjugated anti-IgG and Antibodies against GRP78, Occludin, CHOP, Claudin5, CD31, GAPDH were bought from Santa (Santa Cruz Biotechnology, CA, USA). Alexa Fluor 488-conjugated anti-IgG and Anti-p120-Catenin, XBP-1, β-Catenin, ATF6, Cleaved caspase-12, ATF4, and PDI were bought from Abcam (Abcam, Cambridge, Britain).
6
Western Blot Analysis of Apoptosis Markers
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The transfected cells were collected after 48 h, and RIPA cell lysate was added. Total cell proteins were extracted on ice, and then the protein concentration was detected by a BCA protein detection kit. The protein sample was heated and denaturized in a boiling water bath. The denaturized protein sample of the equal amount was added to the loading hole for SDS-PAGE gel electrophoresis. After the protein was separated, it was transferred to the PVDF membrane for electrotransfer. The transformed membrane was blocked with 5% skimmed milk at room temperature for 1 h. The primary antibodies including cleaved-caspase-3, cleaved-caspase-9, cleaved-caspase-1, cleaved-caspase-12, GRP78, CHOP, IL-1β, N-GSDMD, TGFB1, and β-actin (1:1000, Abcam, Cambridge, Britain) were diluted and incubated overnight at 4°C, and then the secondary antibody (1:3000, Abcam, Cambridge, Britain) was diluted and incubated for 30 min at room temperature, and washed 3 times with TBST. Proteins were detected by the chemiluminescence method, and the relative protein expression of each group was calculated by the ImageJ analysis software (Thermo Fisher Scientific Inc., USA). Analysis on each protein sample was repeated 3 times [19 (link)].
7
Endothelial Cell Culture and Analysis
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DMSO (Sigma-Aldrich) was used to dissolve non-water soluble reagents and as a vehicle control. Endothelial Cell Medium (ECM) which contains 500 ml of basal medium, 25 ml of fetal bovine serum (FBS, Cat. No. 0025), 5 ml of endothelial cell growth supplement (ECGS, Cat. No. 1052) and 5 ml of penicillin/streptomycin solution (P/S, Cat. No. 0503) were purchased from ScienCell Research Laboratories. Antibodies against Occludin, Claudin5, CHOP, GRP78, and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-p62 and microtubule-associated protein-1 light-chain 3 (LC3) were purchased from Cell Signaling Technologies (Danvers, MA, USA). Anti-β-catenin, P120-catenin, cleaved caspase-12, and PDI were purchased from Abcam. All chemicals including RA were from Sigma Chemical Company.
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