Immunohistochemical staining was conducted on 4‐mm sections of heart tissue using Mff 1:500 (Abcam), phospho‐endothelial nitric oxide synthase (p‐eNOS; Ser1177) 1:200 (Abcam), intercellular adhesion molecule–1 1:500 (Abcam), vascular cell adhesion molecule–1 1:500 (Abcam), F4/80 1:500 (Abcam), and plasma albumin 1:500 (Abcam). The primary antibodies were as follows: CD31 (1:1500, Abcam), vascular endothelial cadherin (VE‐cadherin; 1:1000, Abcam), cyt‐c (1:500, Abcam), Mff (1:1000, Abcam), pDrp1 (1:500, Abcam), HK2 (1:500, Abcam), cleaved caspase3 (CL.caspase3; 1:1000, Abcam), and pro‐caspase3 (1:2000, Abcam). 4′,6‐Diamidino‐2‐phenylindole dihydrochloride (Sigma‐Aldrich, USA), lysosome stain, and a mitochondrion‐selective MitoFluor stain (Molecular Probes, USA) were used to marker the nuclear, lysosome, and mitochondria, respectively. For the cross‐linking of VDAC1, treated cells were harvested and added with dimethyl sulfoxide as a vehicle control (2%, as used in compound‐containing samples) or cross‐linked with 0.5 mm ethylene glycolbis (succinimidyl succinate) for 10 minutes at 30°C followed by 20 mm Tris‐HCl (pH 7.4) to quench the reaction. Samples were determined by SDS‐PAGE via immunoblotting.
P drp1
Manufactured by Abcam
Sourced in United States
P-DRP1 is a recombinant protein. It is the phosphorylated form of the Dynamin-related protein 1 (DRP1) enzyme.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole dihydrochloride, by Merck Group (1 mentions)
Cyt c, by Abcam (1 mentions)
Vascular endothelial cadherin ve cadherin, by Abcam (1 mentions)
Mitofluor stain, by Thermo Fisher Scientific (1 mentions)
F4 80, by Abcam (1 mentions)
Pro caspase 3, by Abcam (1 mentions)
Enhanced chemi luminescence (ecl), by Beyotime (1 mentions)
P66shc, by BD (1 mentions)
β actin, by Proteintech (1 mentions)
Cyp2e1, by Proteintech (1 mentions)
Gapdh, by Abcam (1 mentions)
Caspase 3, by Abcam (1 mentions)
Bcl2 associated x (bax), by Abcam (1 mentions)
Coxii, by Abcam (1 mentions)
Cox 4, by Abcam (1 mentions)
Bcl xl, by Abcam (1 mentions)
Cd133, by Aviva Systems Biology (1 mentions)
Cox 1, by Abcam (1 mentions)
Goat anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Goat anti mouse igg antibody, by Santa Cruz Biotechnology (1 mentions)
9 protocols using p drp1
1
Immunohistochemical Analysis of Cardiac Tissue
2
Mitochondrial Dynamics in Liver Tissues
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Liver tissues or AML12 cells were lysed, and protein concentrations were measured. The relative protein expression in liver tissues or AML12 cells was detected by Western blotting. The primary antibodies included antibodies against the following: p66Shc (BD Biosciences, USA, 610878), p-p66Shc (Bioss, China, bs-3410R), CYP2E1 (Proteintech, China, 19937-1-AP), OMA1 (Santa, USA, sc-515788), OPA1 (Proteintech, China, 27733-1-AP), MFN2 (Bioss, China, bs-23685R), DRP1 (Wanleibio, China, WL03028), p-DRP1 (Abcam, USA, ab193216), and beta-actin (Proteintech, China, 60008-1-Ig). Secondary antibodies were obtained from Proteintech. The protein bands were visualized with an enhanced chemiluminescence reagent (ECL, Beyotime, Hangzhou, China).
3
Mitochondrial Dynamics and EV Profiling
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Mitochondria were isolated from PK1 cells using a mitochondria isolation kit (Thermos Fisher, USA), following vendor’s instructions, as we have shown37 . Protein concentration was measured with Bradford Protein Assay. Specific antibodies against Drp1 (1:1000; Abcam), Mfn2 (1:1000; Abcam) and OPA1 (1:500; Abcam) were used with blotting protocols. COXIV (1:5000; Abcam) was used as loading controls. Cell surface, EV or kidney protein expression was studied in homogenate. Specific antibodies against CD9 (1:5,000; Abcam), CD81 (1:1,000; Abcam), CD29(1:1000, Abcam), CD24(1:500, Abcam), CD133(1:500, Aviva Systems Biology), COX I (1:1000, Abcam), COX II (1:1000,Abcam), Caspase3 (1:1000, Abcam), Bax(1:1000,Abcam), Bcl-XL (1:1000, Abcam), COX IV, Drp1, p-Drp1(1:1000, Abcam) antibodies were used with blotting protocols, and GAPDH (1:1000, Abcam) as loading controls.The density of each band was analyzed by Image-Pro Plus 6.0 software.
4
Western Blot Analysis of MSCs
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Proteins (30 μg) in the cell lysate of healthy-MSCs and CKD-MSCs were resolved by 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The resolved proteins were transferred to an 8 μm pore size nitrocellulose membrane. The membrane was blocked using 5% skim milk prepared in TBST (10 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.05% (v/v) Tween 20) for 1 h at room temperature. The membrane was then treated with primary antibodies. The antibodies used were directed against p-PERK, anti-PERK, p-JNK, JNK, MFN1, PrPC, and β-actin (all 1:300 dilution, all from Santa Cruz Biotechnology, Dallas, TX, USA), p-eIF2α, eIF2α, ATF4, p-DRP1 (both 1:1000 dilution, both from Abcam, Cambridge, UK), p-IRE1α, IRE1α, CHOP, OPA1, and PGC-1α (all 1:1000 dilution, all from Novus, Centennial, CO, USA). Next, each membrane was washed twice, and the primary antibodies were detected using goat anti-rabbit IgG or goat anti-mouse IgG antibodies (Santa Cruz Biotechnology). The protein bands were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Little Chalfont, UK).
5
Adiponectin Signaling Pathway Analysis
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Recombinant mouse gCTRP9 protein was purchased from Aviscera Bioscience (Santa Clara, CA). Antibodies for Glut1, ABCA1, PPAR-y, HIF-1a, AdipoR1, actin, tubulin was obtained from Abcam, Drp1, p-Drp1 (Ser616), Erk1/2, p-Erk1/2(Thr202/Tyr204), Jnk, p-Jnk (Thr183/Tyr185), p38, p-p38 (Thr180/Tyr182), Opa1, Mff, Mfn-2, Ucp2 were bought from CST. U0126, SP600125 and SB203580 were obtained from MCE. Cell culture medium was purchased from HyClone Laboratories (Utah, USA).
6
Murine Hippocampal Neuronal HT22 Cell Culture
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Murine hippocampal neuronal HT22 cells (originally purchased from ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM)/F12 (Invitrogen, Carlsbad, CA), supplemented with 10% fetal bovine serum (FBS, HyClone), 100 nM penicillin/streptomycin, at 37 °C in 5% CO2 and 90% relative humidity. The culture medium was renewed every 2 days. Sodium glutamate, and sodium selenite (Sigma-Aldrich, St Lois, MO), were reconstituted in water and diluted to appropriate concentrations in cell culture medium. Antibodies directed against caspase-3, caspase-9, apoptosis protease-activating factor-1 (Apaf-1), cleaved caspase-3, cleaved caspase-9 were purchased from Cell Signaling Technology, Danvers, MA. Anti LC3A/B, Fis1, Drp1, p-Drp1, and β-actin were purchased from Abcam, Cambridge, MA.
7
Apoptosis and Mitochondrial Regulation in H9c2 Cells
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Western blotting was performed to detect apoptosis-related, mitochondrial function-related, and extracellular signal-regulated kinase (ERK) protein expression in H9c2 cells. Total protein was extracted from H9c2 cells using radioimmunoprecipitation assay lysis buffer (Solarbio), and protein concentration was quantified using a bicinchoninic acid assay kit (Solarbio). Twenty micrograms of proteins from each group were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. After blocking with 5% skim milk, the membranes were incubated for 1 h with primary antibodies against dynamin-related protein 1 (DRP1), mitofusin 2 (MFN2), phosphorylation (p)-DRP1-S637, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X (Bax), cleaved caspase-3, ERK1/2, p-ERK1/2, and β-actin (internal reference), followed by 1 h of incubation with the goat anti-rabbit IgG secondary antibody. All antibodies were purchased from Bioswamp, except for cleaved caspase-3 (Abcam, Cambridge, UK), p-ERK1/2 (Abcam), and p-DRP1 (CST).
8
Western Blot Analysis of OXPHOS and Mitochondrial Dynamics
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Western blot was conducted as per our previous work [18 (link)]. Aliquots of cytoplasmic or mitochondrial extracts were fractionated by electrophoresis on 8–12% SDS polyacrylamide gels (BioRad, Hercules, CA). Subsequently, proteins were transferred to PVDF membranes and blots were blocked with 1% casein (w/v) nonfat milk dissolved in Tris-buffered saline (TBS) with Tween-20 (BioRad, Hercules, CA). Total OXPHOS cocktail, DRP-1, pDRP-1, and MFN-2 primary antibodies were obtained from Abcam (Cambridge, UK). . All blots shown are representative samples from 3 to 7 experiments.
9
Protein Markers in Brain Injury
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