Whole-cell lysates were prepared from the wild-type KATOIII strain and its derived clones B3 and B4 using a Total Protein Extraction Kit (TaKaRa). Supernatant was prepared after the cells had been inoculated at 7 × 105/ml into serum-free medium 72 h prior to harvesting, followed by concentration with Amicon® Ultra-10K (Merck Millipore, Burlington, MA). Western blotting was carried out with rabbit anti-OBP2B monoclonal antibody (MA5-30722; Thermo Fisher Scientific) or anti-β-actin monoclonal antibody (017-24551; Wako, Osaka, Japan), followed by treatment with Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, MA) and densitometry measurements with a LAS-3000 and MultiGauge v3.0 (FujiFilm, Tokyo, Japan).
Anti β actin monoclonal antibody
Manufactured by Fujifilm
Sourced in Japan
The Anti-β-actin monoclonal antibody is a laboratory reagent used to detect and quantify the presence of the beta-actin protein in biological samples. It is a highly specific and sensitive tool for researchers studying cell structure, function, and dynamics.
Automatically generated - may contain errors
Lab products found in correlation
Total protein extraction kit, by Takara Bio (1 mentions)
Amicon ultra 10k, by Merck Group (1 mentions)
Amersham ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Multi gauge v3, by Fujifilm (1 mentions)
Las 3000, by Fujifilm (1 mentions)
Mitotracker, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Iron 3 chloride hexahydrate, by Fujifilm (1 mentions)
Celltracker green cmfda, by Thermo Fisher Scientific (1 mentions)
Lysotracker, by Thermo Fisher Scientific (1 mentions)
Can get signal solution 1, by Toyobo (1 mentions)
Il 1β antibody, by Cell Signaling Technology (1 mentions)
Tnf α antibody, by Cell Signaling Technology (1 mentions)
Nf κb antibody, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit antibody, by Vector Laboratories (1 mentions)
Phospho nf κb antibody, by Cell Signaling Technology (1 mentions)
Immun blot pvdf membrane, by Bio-Rad (1 mentions)
3 protocols using anti β actin monoclonal antibody
1
Quantitative Western Blot Analysis
2
Synthesis and Characterization of HMRhoNox-M
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HMRhoNox-M was prepared according to the reported procedure [24] (link). We purchased iron (III) chloride hexahydrate (Wako, Osaka, Japan), LysoTracker (Molecular Probes, Eugene, OR), MitoTracker (Molecular Probes), CellTracker Green CMFDA (Molecular Probes), Hoechst 33342 (Molecular Probes), Cellmatrix Type I-P collagen (Nitta Gelatin, Inc., Osaka, Japan), anti-human transferrin receptor antibody (Zymed, 13-6800; San Francisco, CA), anti-ferroportin 1 antibody (Novus Biologicals, NBP1-21502; Littleton, CO), anti-CD10 monoclonal antibody (Novocastra, NCL-L-CD10-270; Newcastle, UK), anti β-actin monoclonal antibody (Wako, 011-24554; Tokyo, Japan), Stealth siRNAs for human transferrin receptor 1 (TfR1) (HSS110674, HSS110676 and HSS186305) and a non-targeting siRNA (HSS12935-112) as a negative control (Invitrogen, Carlsbad, CA). We prepared ferric nitrilotriacetate (Fe(III)-NTA) solution by mixing 300 mM ferric nitrate and 600 mM nitrilotriacetate, followed by pH adjustment to 7.4 with sodium hydrogen bicarbonate as described [28] (link). The other chemicals were of analytical grade.
3
Western Blot Analysis of Inflammatory Markers
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Liver protein samples were separated by electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto Immun-Blot PVDF membranes (Bio-Rad Laboratories, K.K., Tokyo, Japan). The membranes were then incubated overnight at 4 °C with IL-1β antibody (#12242; Cell Signaling,), TNF-α antibody (#11948; Cell Signaling), NF-κB antibody (#8242; Cell Signaling), phospho-NF-κB antibody (#3033; Cell Signaling) and anti-β-actin monoclonal antibody (Wako Pure Chemical Co., Osaka, Japan) diluted in Can Get Signal solution 1 (Toyobo, Osaka, Japan), after which the membranes were incubated for 1 h at room temperature with a horseradish peroxidase-conjugated goat anti-rabbit antibody (Vector Laboratories, Burlingame, CA, USA).
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