The canines were subjected to electrophysiological testing after seven days of continuous rapid pacing and then were anesthetized and executed, followed by the sampling of atrial tissue. Paraffin embedding was performed after fixation using paraformaldehyde. For histological analysis, canine atrial tissues were sectioned into 5 μM thick slices. The sections were then stained with hematoxylin and eosin and Masson's trichrome to assess inflammatory infiltration and collagen deposition, respectively. To determine the expression of KCa3.1, CD68, CD81, cTnI, iNOS, and Rab27a, the sections were incubated with KCa3.1 (Proteintech, China; 1 : 100), CD68 (Servicebio, China; 1 : 200), CD81 (Proteintech, China; 1 : 200), cTnI (Affinity, China; 1 : 200), iNOS (Servicebio, China; 1 : 200), and Rab27a antibodies (Proteintech, China; 1 : 300). Immunohistochemistry and immunofluorescence images were captured with a fluoroscope and analyzed by ImageJ.
Rab27a
Manufactured by Proteintech
Sourced in United States, China
Rab27a is a member of the Rab family of small GTPases. It functions as a molecular switch that cycles between an active GTP-bound state and an inactive GDP-bound state. Rab27a plays a role in the regulation of vesicle trafficking and exocytosis in various cell types.
Automatically generated - may contain errors
Lab products found in correlation
Image quant las 300, by GE Healthcare (3 mentions)
P erk, by Cell Signaling Technology (3 mentions)
Gapdh, by Proteintech (3 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
Calnexin, by Proteintech (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (2 mentions)
Nsmase2, by Santa Cruz Biotechnology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bcl 2 15071, by Cell Signaling Technology (1 mentions)
Cleaved parp, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Chemiluminescence reagent, by Bio-Rad (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
Nsmase, by Abcam (1 mentions)
Pd l1, by Cell Signaling Technology (1 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
Sds lysis buffer, by Beyotime (1 mentions)
Irak1, by Santa Cruz Biotechnology (1 mentions)
11 protocols using rab27a
1
Canine Atrial Tissue Analysis
2
Western Blot Analysis of Cellular Proteins
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Total proteins from cells were extracted in cell-lysis buffer (Pierce, Rockford, IL) and quantified using the Bradford method. About 50 μg protein was transferred to PVDF membranes after separated by SDS-PAGE. The membranes were incubated at 4°C overnight with Rab27A (1:800, Proteintech, USA), cyclin E (4129), cyclin D1 (2978), p-IκB (2859), IκB (4814), p-p65 (3033), p65 (8242), caspase 3 (9668), cleaved caspase 3 (9664), PARP (9532), cleaved PARP (5625), cytochrome c (11940), Bcl-2 (15071) (1:1000; Cell signaling, Boston, USA) and GAPDH (1:2000; Santa Cruz, USA). After incubation with HRP-coupled anti-mouse/rabbit IgG antibody (1:2000 dilution, Cell Signaling Technology, USA) at 37°C for 2 hours. Target proteins on PVDF membrane were visualized using ECL (Pierce, USA) and captured using a DNR BioImaging System (DNR, Israel).
3
Oligonucleotide Primers and Immunoblot Analysis
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Oligonucleotide primers for convention PCR was synthesized by Integrated DNA technologies (Coralville, Iowa) and are listed in Table S1 . Total RNA and proteins were isolated from various using standard protocols as we described11 (link). Total RNA was subjected to conventional PCR analyses using a master mix from Bio-Rad Laboratories (Hercules, CA). Protein extracts were subjected to immunoblot analysis using antibodies against PD-L1, EGFR, Alix, p-ERK, ERK, p-JNK, JNK, p-p38, p38, cyclin D1 (Cell Signaling Technology, Danvers, MA, USA), CD63, Her2 and GAPDH (Santa Cruz Biotechnology, Dallas, TX), Rab27a (Proteintech, Chicago, IL, USA) and nSMase (Abcam, San Francisco, CA, USA). Immune complexes were detected with appropriate secondary antibodies from Jackson ImminoResearch Inc. (West Grove, PA, USA) and chemiluminescence reagents (Bio-Rad, Hercules, CA, USA) as described13 (link),44 (link). Immunoblot signals were captured using the Image Quant Las 300 (GE Healthcare, Piscataway, NJ, USA). Densitometric analysis was performed using ImageJ (NIH, Bethesda, MD, USA, http://imagej.nih.gov/ij/ ).
4
Exosome Protein Characterization Assay
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Cells were lysed in 200 μl SDS lysis buffer (Beyotime, Shanghai, China) with 1% PMSF (Beyotime, Shanghai, China) and boiled at 100 °C for 15 min. Forty micrograms of each protein sample was separated by electrophoresis with 12% polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, CA, USA). Thereafter, according to the manufacturer’s instructions, the proteins were incubated with appropriate primary antibodies and secondary antibodies. Primary antibodies of Alix, CD63, CD81, Calnexin, RAB27A, and the loading control (GAPDH) were purchased from Proteintech Group. Results were analyzed by ImageJ version 1.47 (National Institutes of Health, Maryland, USA). Three independent experiments were done for statistical analysis.
5
Western Blot Analysis of Viral and Cellular Proteins
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Western blot assay was carried out as follow: cells were lysed with RIPA buffer (Santa Cruz, USA) and cleared lysate was collected by centrifugation for protein separation on 10% SDS-polyacrylamide gel. The proteins were transferred onto PVDF membranes (Millipore) and detected with respective antibodies at 4°C overnight, followed by incubation with either IRDye Fluor 680-labeled IgG or IRDye Fluor 800-labeled IgG secondary antibody (Li-Cor Bioscience). The images were scanned and quantified by densitometric analysis by Li-COR Odyssey Infrared Imager. Primary antibodies against VP1 (Millipore), CD81 (Cell Signaling Technology), IRAK1 (Santa Cruz), CD63 (Abcam), ISG15 (Abcam) and Rab27a, TRAF6, STAT1, TSG101, BST-2/Tetherin, and GAPDH (all from Proteintech), VP2, 3AB, 3C and 3D (all from Genetex) were used.
6
Exosomal Protein Extraction and Analysis
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Total cell proteins were extracted by RIPA (Solarbio, China). Exosomal proteins were extracted by repeated freeze–thaw lysis at -80℃. Cellular and exosomal protein concentrations were measured by BCA protein assay kit (CWBIO, China) and micro-BCA protein assay kit, respectively. Each protein sample (20 μg) was separated by electrophoresis with 12% polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, USA). Subsequently, PVDF membranes were blocked, incubated with antibodies and washed. Primary antibodies of Alix, CD63, CD81, Calnexin, RAB27a, and the loading controls (GAPDH and β-Actin) were purchased from Proteintech Group, USA. LC3, P62 and TSG101 antibody was purchased from Abcam, Britain. The secondary Antibodies: anti-Rabit and anti-Mouse was purchased from Proteintech Group.
7
Immunoblot Analysis of Exosome Biogenesis
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Protein extracts were subjected to immunoblot analysis using antibodies against Alix, p-ERK, ERK (Cell Signaling Technology, Danvers, MA, USA), GAPDH (Santa Cruz Biotechnology, Dallas, TX), Rab27a (Proteintech, Chicago, IL, USA) and nSMase2 (Santa Cruz Biotechnology). Immune complexes were detected with appropriate secondary antibodies from Santa Cruz Biotechnology and chemiluminescence reagents (Pierce, Rockford, IL, USA) as we described54 (link). Immunoblot signals were captured using the Image Quant Las 300 (GE Healthcare, Piscataway, NJ, USA). Densitometric analysis was performed using ImageJ (NIH, Bethesda, MD, USA, http://imagej.nih.gov/ij/ ).
8
Optimized Protein and RNA Extraction Protocol
9
Exosome Protein Characterization by Immunoblotting
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Exosome proteins or cell lysates were blotted on a nitrocellulose membrane (5 μg) and were blocked in 5% nonfat dry milk prepared in 0.2% Tween-20 and 1× Tris Buffer Saline (TBS) and were subjected to immunoblot analysis using antibodies against Flotillin-1 (1:250, Thermo Fisher Scientific, MA, USA), TSG101 (1:250, Invitrogen, Thermo Fisher Scientific, MA, USA), ALIX, (1: 250, Cell Signaling Technology, Danvers, MA, USA), CD63, (1: 250, Cell Signaling Technology, Danvers, MA, USA), GAPDH (1: 250, DSHB, Iowa City, IA, USA), and Rab27a (1: 250, Proteintech, Chicago, IL, USA). Immune complexes were detected with appropriate goat anti-mouse (1:1000, Cell Signaling Technology, Danvers, MA, USA) and goat anti-rabbit (1:1000, Santa Cruz Biotechnology, Texas) secondary antibodies, the signal was detected using SuperSignal West Femto Maximum Sensitivity Substrate and imaged using Bio-Rad ChemiDoc XRS + System (Bio-Rad Laboratories, Hercules, CA, USA) as we described [6 ,24 ]. The Immunoblot signal was captured and analyzed via densitometric analysis on ImageJ software (NIH, Bethesda, MD, USA).
10
Western Blotting Analysis of Cellular Proteins
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For Western blotting analysis, cells were lysed in lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 10% glycerol, 0.1% Tween20, 10 mM β-glycerophosphate) with 1% Protease inhibitor cocktail (Nacalai Tesque). The protein concentration was determined using a Pierce™ BCA Protein Assay Kit (23225, Thermo Fisher Scientific), and the proteins were transferred to a PVDF membrane (EMD Millipore) after SDS-PAGE. Primary antibodies used in this study were anti-SMS2 (sc-366682, Santa Cruz), nSMase2 (sc-166637, Santa Cruz), H-Ras (sc-29, Santa Cruz), Alix (12422, Proteintech, Rosemont, IL, USA), Rab27a (17817, Proteintech), Caspase3 (9662, Cell Signaling Technology), Caspase1 (sc-1780, Santa Cruz), ATG5 (12994, Cell Signaling Technology), p62 (PM045, MBL International, Woburn, MA, USA), LC3 (2775, Cell Signal Transduction), α-tubulin (T9026, Sigma-Aldrich). After incubation with secondary antibodies (GE Healthcare, Chicago, IL, USA), membranes were treated with the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific), and detected by FUSION SOLO S (Vilber Lourmat, Collegien, France).
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