Under normal conditions, proteins in the lysates of the cells treated with CoCl2 and/or oligomycin A were subjected to 8.5 or 12.5% SDS-PAGE. After electrophoresis, the proteins were transferred to PVDF membranes (Immobilon-FL, Merck-Millipore, Cork, Ireland) . The membrane was blocked with 5% skim milk (Snow Brand, Japan) and incubated with the primary antibodies overnight at 4 °C followed by incubation with the suitable secondary antibody. The primary antibodies used were anti-Oct-4A (9B7) mouse monoclonal IgG (1: 1000, #4286, Cell Signaling Technology, MA), anti-HIF-1α mouse monoclonal IgG1 clone # 241,809 (1: 1000, R&D Systems, MN), anti-CD31 rabbit polyclonal antibody (1:500, ab28364, Abcam, UK), anti-mouse β-tubulin rabbit polyclonal antibody (1: 1000, #2146 s, Cell Signaling Technology), anti β-actin mouse monoclonal antibody (1:2000, 010–27,841, Wako) and anti-GFP goat polyclonal antibody labeled with HRP (1: 2000, ab6663, Abcam, UK). The secondary antibodies used were anti-rabbit IgG goat IgG linked with HRP (1:10,000, #7074 s, Cell Signaling Technology)and anti-mouse IgG goat IgG linked with HRP (1: 10,000, #7076 s, Cell Signaling Technology). HRP reaction was performed with Ez West Lumi plus (ATTO, Japan), and the developed fluorescence intensity was detected by Light Capture II (ATTO, Japan) or Lumino Graph I (ATTO, Japan).
Anti β actin mouse monoclonal antibody
Manufactured by Fujifilm
Sourced in Japan, United States
The Anti-β-actin mouse monoclonal antibody is a laboratory reagent used for the detection and quantification of the β-actin protein in biological samples. This antibody is a specific and sensitive tool for researchers studying cellular structure and function.
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Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Mouse monoclonal anti mt 1 2 antibody e9, by Agilent Technologies (2 mentions)
Bovine aortic endothelial cells, by Cell Applications (2 mentions)
Geneace sybr qpcr mix α, by Nippon Gene (2 mentions)
Anti cd31 rabbit polyclonal antibody, by Abcam (1 mentions)
Immobilon fl, by Merck Group (1 mentions)
Fluorescein isothiocyanate isomer 1, by Merck Group (1 mentions)
N 5 amino 1 carboxypentyl iminodiacetic acid ab nta, by Dojindo Laboratories (1 mentions)
Oleylamine, by Fujifilm (1 mentions)
Imobiron, by Merck Group (1 mentions)
May gr nwald and giemsa stain solution, by Merck Group (1 mentions)
Cmf pbs, by Nissui Pharmaceutical (1 mentions)
Horseradish peroxidase conjugated anti mouse igg antibody, by Cell Signaling Technology (1 mentions)
Qiazol lysis reagent, by Qiagen (1 mentions)
Bicinchoninic acid protein assay reagent kit, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Nissui Pharmaceutical (1 mentions)
Calcium and magnesium free phosphate buffered saline, by Nissui Pharmaceutical (1 mentions)
Prl sv40, by Promega (1 mentions)
Chemi lumi one l, by Nacalai Tesque (1 mentions)
Fetal bovine serum (fbs), by Cytiva (1 mentions)
8 protocols using anti β actin mouse monoclonal antibody
1
Western Blot Analysis of Stem Cell Markers
2
Fluorescent Nanoparticle Antibody Labeling
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Poly(D,L-lactide-co-glycolic) acid (PLGA, 85:15, MW: 190,000–240,000) and fluorescein isothiocyanate isomer I were purchased from Sigma-Aldrich (St. Louis, MO, USA). N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (water-soluble carbodiimide, WSC), and N-(5-amino-1-carboxypentyl) iminodiacetic acid (AB-NTA) were from DOJINDO (Kumamoto, Japan). A purified mouse anti-BiP/GRP78 antibody (Cat#610979) was from BD Transduction Laboratories™ (Franklin Lakes, NJ, USA). Oleylamine and an anti-β-actin mouse monoclonal antibody were purchased from FUJIFILM Wako Pure Chemical (Osaka, Japan). Anti-mouse immunoglobulin G (IgG), horseradish peroxidase (HRP)-linked (#7076) and anti-rabbit IgG, HRP-linked (#7074), anti-CHOP (#2895), anti-poly(ADP-ribose) polymerases (PARP) (#9542), anti-cleaved PARP (#5625), anti-cleaved cysteine aspartate-specific protease (caspase) 7 (#9491), anti-cleaved caspase 3 (#9661), anti-phospho-eIF2α (#3398), and anti-total eIF2α (#5324) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). An anti-iNOS mouse monoclonal antibody [37 (link)] and anti-SubAB anti-serum [38 (link)] were prepared as reported previously.
3
Ceramide Regulation Pathway Analysis
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The ceramide analysis performed using thin-layer membranes were incubated with anti-CerS3 rabbit antibody (1:2,000; Cusabio, Houston, TX, USA), with anti-ELOVL4 rabbit antibody (1:1,000; Cusabio, USA), or with anti-β-actin mouse monoclonal antibody (1:5,000, Wako Chemical, Tokyo, Japan). This was followed by incubation with horseradish peroxidase-conjugated antirabbit immunoglobulin G (IgG) goat antibody (1:5,000; BioSource, Camarillo, CA, USA) for CerS3 or ELOVL4, or horseradish peroxidase-conjugated anti-mouse IgG goat antibody (1:5,000; Wako Chemical) for β-actin. The blots were subsequently developed using an Immunostar LD (Wako Chemical, Japan) or Imobiron (Merck Millipore, Billerica, MA, USA) using LuminoGraph (Atto Corporation, Tokyo, Japan). The bands were densitometrically analyzed using ImageJ (National Institutes of Health, Bethesda, MD, USA).
4
Bovine Aortic Endothelial Cell Culture
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Bovine aortic endothelial cells were purchased from Cell Applications (San Diego, CA, USA). The following materials were purchased from the respective vendors: Dulbecco's modified Eagle's medium (DMEM) and calcium-and magnesium-free phosphate-buffered saline (CMF-PBS; Nissui Pharmaceutical, Tokyo, Japan); fetal bovine serum and a High-Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific, Waltham, MA, USA); QIAzol lysis reagent (QIAGEN, Valencia, CA, USA); GeneAce SYBR qPCR Mixα (Nippon Gene, Tokyo, Japan); mouse monoclonal anti-MT-1/2 antibody (E9; Dako, Glostrup, Denmark); Mouse monoclonal anti-β-actin antibody (Wako Pure Chemical Industries, Osaka, Japan); horseradish peroxidase-conjugated antimouse IgG antibody (#7076; Cell Signaling, Beverly, MA, USA); May-Grünwald and Giemsa stain solution (Merck KGaA, Darmstadt, Germany); and cadmium chloride, manganese chloride tetrahydrate, and other reagents (Nacalai Tesque, Kyoto, Japan).
5
Evaluation of Nrf2 Activation in Endothelial Cells
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Cu10 was purchased from Tokyo Chemical Industry (Tokyo, Japan). Bovine aortic endothelial cells were purchased from Cell Applications (San Diego, CA, USA). Dulbecco's modified Eagle's medium and calcium-and magnesium-free phosphate buffered saline were from Nissui Pharmaceutical (Tokyo, Japan). Fetal bovine serum was from HyClone Laboratories (Waltham, MA, USA). Bicinchoninic acid protein assay reagent kit was from Thermo Fisher Scientific (Waltham, MA, USA). Polyvinylidene difluoride membrane (0.2 μm), sodium diethyldithiocarbamate trihydrate (Na01), and mouse monoclonal anti β-actin antibody were from Wako Pure Chemical Industries (Osaka, Japan). Mouse monoclonal anti-MT-1/2 antibody (E9) was from Dako (Glostrup, Denmark). Rabbit polyclonal anti-human Nrf2 antibody (H-300) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroxidase-conjugated anti-rabbit IgG antibody (#7074) and horseradish peroxidase conjugated anti-mouse IgG antibody (#7076) were from Cell Signaling (Beverly, MA, USA). High-Capacity cDNA Reverse Transcription kit was from Applied Biosystems (Foster, CA, USA). Gene Ace SYBR qPCR Mixα was from Nippon Gene (Tokyo, Japan). Dual-Luciferase Reporter Assay System and pRL-SV40 were from Promega (Madison, WI, USA). Chemi-Lumi One L and other reagents were from Nacalai Tesque (Kyoto, Japan).
6
Western Blot Analysis of Protein Samples
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We reduced the cell extracts and liver homogenate samples with mercaptoethanol at 95 °C for 10 min. We loaded the samples at 10 μg of protein per lane and then separated them on a 12% polyacrylamide gel. Following the electrophoresis, we blotted the proteins on a nitrocellulose membrane (ClearTrans Nitrocellulose Membrane; Fujifilm Wako Pure Chemical) in a semidry blotting system (NA‐1512S; Nihon Eido, Tokyo, Japan) [20 (link)]. The nitrocellulose membranes were blocked with 2% skim milk. We then incubated the membranes with rabbit anti‐CA3 antibody (1 : 8000; Proteintech, Rosemont, IL, USA), rabbit anti‐catalase antibody (1 : 10,000; produced by our laboratory), mouse anti‐β actin monoclonal antibody (1 : 10,000; Fujifilm Wako Pure Chemical), or mouse anti‐glyceraldehyde‐3‐phosphate dehydrogenase monoclonal antibody (1 : 5000; Fujifilm Wako Pure Chemical). This was followed by incubation with horseradish peroxidase (HRP)‐conjugated goat anti‐rabbit immunoglobulin G (IgG) antibody (1 : 10,000; SeraCare, Milford, MA, USA) or HRP‐conjugated goat anti‐mouse IgG antibody (1 : 2500; Biosource, Camarillo, CA, USA). Finally, we visualized the protein bands using ImmunoStar LD or ImmunoStar Zeta (both Fujifilm Wako Pure Chemical) with a LuminoGraph (Atto, Tokyo, Japan). We subjected the bands to densitometric analysis using imagej (National Institutes of Health, Bethesda, MD, USA).
7
Quantifying ACE2 Expression in KATOIII and NUGC-4 Cells
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Whole-cell lysates were prepared from KATOIII and NUGC-4 cells incubated with 0, 25 or 50 nM panobinostat for 24 or 48 h. One hundred or 40 µg of total protein was applied to each lane for the KATOIII or NUGC-4 assays, respectively. The transferred membrane was reacted with a recombinant rabbit anti-ACE2 monoclonal antibody (#ab108252, Abcam), followed by treatment with anti-rabbit immunoglobulins/HRP (#P0448, DakoCytomation) and Amersham ECL Prime Western Blotting Detection Reagent (#RPM2232, GE Healthcare). Then, densitometry measurements were conducted with a LAS-3000 and MultiGauge v3.0 (FujiFilm, Tokyo, Japan). Subsequently, the same membrane was rinsed, processed with blocking buffer, reacted with mouse anti-β-actin monoclonal antibody (#017-24551, Wako) and anti-mouse IgG (H + L)-HRP conjugate (#172-1011, BioRad). Detection and densitometry measurements were performed again as described above.
8
SARS-CoV-2 Spike Protein Detection by Western Blot
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Transfected cells were lysed on ice for 15 min in a buffer (100 mM NaCl, 1 mM TCEP [Tris (2-carboxyethyl) phosphine hydrochloride], 2× protease inhibitor, and 10 mM HEPES; pH 7.5) containing 1% n-dodecyl-β-D-maltoside (DDM; Thermo Scientific). The resultant samples were resuspended in 1× Laemmli buffer containing 5% β-mercaptoethanol (Bio-Rad), boiled for 10 min, and subjected to protein separation by SDS-PAGE in 4–20% Mini-PROTEAN TGX precast gels (Bio-Rad) before transferred to nitrocellulose membranes (Wako). The membranes were incubated in a blocking buffer (Nacalai Tesque) for 1 h at room temperature and then mixed with primary antibodies, including rabbit anti-SARS-CoV-2 Spike (S1/S2) polyclonal antibody (1:2000; Invitrogen, Cat# PA5-112048) and mouse anti-β-actin monoclonal antibody (1:5,000; Wako, Cat# 010-27841), followed by staining with the horseradish peroxidase (HRP)-conjugated anti-rabbit (1:50,000; GE healthcare, Cat# NA934VS) and anti-mouse (1:25,000; GE healthcare, Cat# NA931VS) IgG secondary antibodies. The membrane was developed with the ImmunoStar LD enhanced chemiluminescence reagents (Wako) and visualized using ImageQuant LAS 4000 (GE Healthcare).
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