Cells and mouse lungs were lysed in a buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100 and 1 mM phenylmethylsulfonyl fluoride and were subjected to Western blot analyses as described in our previous report (Koma et al., 2008 (link)). The recovered bronchoalveolar lavage fluid was directly separated on SDS-PAGE gels. Two kinds of anti-CADM1 antibodies were used, which we previously generated; rabbit polyclonal antibody against the C-terminal peptide (eggqnnseekkeyfi) to detect full-length CADM1 and αCTF (Mimae et al., 2014 (link)), and chicken monoclonal antibody against the ectodomain (3E1) to detect the N-terminal fragment (NTF) released from the cell by CADM1 shedding (Furuno et al., 2005 (link); Nagara et al., 2012 (link)). Other primary antibodies used in this study targeted ADAM10 (rabbit polyclonal; Millipore, Billerica, MA, USA), αSMA (mouse monoclonal clone 1A4; Dako, Santa Clara, CA, USA), E-cadherin (clone 36; BD Bioscience, San Jose, CA, USA), pan-cytokeratin (mouse monoclonal AE1/AE3; Dako), and β-actin (Medical & Biological Laboratories). Peroxidase-conjugated secondary antibodies were purchased from Amersham (Buckinghamshire, England). Immunoreactive band intensities were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA), as described previously (Mimae et al., 2012 (link)).
β actin
Manufactured by Medical & Biological Laboratories
Sourced in United States, Japan
β-actin is a fundamental cytoskeletal protein found in all eukaryotic cells. It is involved in various cellular processes, including cell motility, structure, and division. β-actin serves as a widely used internal control and reference gene in molecular biology and cell biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
α sma, by Agilent Technologies (1 mentions)
Pan cytokeratin, by Agilent Technologies (1 mentions)
Adam10, by Merck Group (1 mentions)
E cadherin, by BD (1 mentions)
Protease inhibitor cocktail, by Promega (1 mentions)
Phosphatase inhibitor cocktail, by Fujifilm (1 mentions)
Enhanced chemoluminescence, by PerkinElmer (1 mentions)
Phosphorylated erk1 2, by Cell Signaling Technology (1 mentions)
Dusp1, by Merck Group (1 mentions)
Bca reagent, by Thermo Fisher Scientific (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
Phosphorylated akt, by Cell Signaling Technology (1 mentions)
P erk 4370, by Cell Signaling Technology (1 mentions)
Erk 4695, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 conjugated donkey anti mouse, by Santa Cruz Biotechnology (1 mentions)
594 conjugated donkey anti rabbit antibodies, by Santa Cruz Biotechnology (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Mcl 1, by Santa Cruz Biotechnology (1 mentions)
8 protocols using β actin
1
CADM1 Shedding and Lung Protein Analysis
2
Western Blot Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell extracts were lysed in SDS buffer, a protease inhibitor cocktail (Promega) and phosphatase inhibitor cocktail (FUJIFILM Wako Pure Chemical Corporation). After quantifying protein concentrations using BCA reagent (Thermo Fisher Scientific), proteins were resolved by SDS‐PAGE and transferred to nitrocellulose membrane by electroblotting. Membranes were blocked in TBS containing 5% skim milk and probed with the following primary antibodies: goat anti‐galectin‐1 antibody (1:1000, R&D systems), rabbit antibodies against ATF2 (1:1000), phosphorylated ATF2 (1:1000), c‐Fos, phosphorylated c‐Fos (1:1000), c‐Jun (1:1000), phosphorylated c‐Jun (1:1000), AKT (also known as protein kinase B) (1:1000), phosphorylated AKT (1:1000), ERK1/2 (1:1000), phosphorylated ERK1/2 (1:1000, Cell Signaling Technology), DUSP1 (1:1000, Millipore) and β‐actin (1:4000, Medical & Biological Laboratories). Horseradish peroxidase‐conjugated anti‐goat and anti‐rabbit IgGs (1:4000, Jackson ImmunoResearch Laboratories) were used as a secondary antibody for chemoluminescence detection. Signals were obtained by enhanced chemoluminescence (Perkin Elmer).
3
Antibody and Inhibitor Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies against Flag (catalog number: M185-3 L) and β-actin (M177–3) were purchased from Medical Biological Laboratories (Nagoya, Japan). Antibodies against matrix metalloproteinase (MMP)9 (A0289) were obtained from ABclonal (Woburn, MA, USA). Antibodies against P21 (2947), cyclin D1 (2978), c-Jun N-terminal kinase (JNK; 9252), phosphorylated (p)-JNK (4668), protein kinase B (AKT; 4691), p-AKT (4060), p38 (9212), p-p38 (4511), extracellular signal-regulated kinase (ERK; 4695) and p-ERK (4370) were purchased from Cell Signaling Technology (Danvers, MA, USA).
The JNK inhibitor JNK-IN-8 (HY-13319, MCE, USA) was dissolved in dimethyl sulfoxide (DMSO) and diluted into a 0.5-μM working solution with complete culture medium, and the same amount of DMSO was set as the control.
The JNK inhibitor JNK-IN-8 (HY-13319, MCE, USA) was dissolved in dimethyl sulfoxide (DMSO) and diluted into a 0.5-μM working solution with complete culture medium, and the same amount of DMSO was set as the control.
4
PDCoV Isolation and Propagation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK-293T cells were cultured and maintained in RPMI-1640 (HyClone, UT), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (PAN-biotech, Bavaria, Germany) at 37 °C in a humidified 5% CO2 incubator. The PDCoV strain CHN-HN-2014 (GenBank accession number KT336560) used in this study was isolated from a suckling piglet with acute diarrhea in China in 2014 [26 (link)]. LLC-PK1 cells, a porcine kidney cell line purchased from the ATCC, were cultured at 37 °C in 5% CO2 in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% heat-inactivated FBS and were used to amplify PDCoV. SEV was acquired from the Centre of Virus Resource and Information at the Wuhan Institute of Virology. Recombinant VSV-GFP was generously provided by Prof. Zhigao Bu at Harbin Veterinary Research Institute, China. Poly(I) and poly(C)-agaroses were bought from Sigma-Aldrich (MO, USA). Mouse monoclonal antibodies against Flag, HA, Myc and β-actin were purchased from Medical & Biological Laboratories (Nagoya, Japan). The PDCoV-N-protein-specific monoclonal antibody was produced in our laboratory as described previously [27 (link)]. Rabbit polyclonal antibodies directed against Flag were bought from ABclone (Wuhan, China). Alexa Fluor 488-conjugated donkey anti-mouse and 594-conjugated donkey anti-rabbit antibodies were purchased from Santa Cruz Biotechnology (CA, United States).
5
Western Blot Analysis of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lysate extracted from a total of 1 × 105 cells was used to perform Western blots analysis. Primary antibodies against pEGFR (1:3000; Cell Signaling Technology, Danvers, MA, USA, 3777 S), EGFR (1:3000; Cell Signaling Technology, 4267 S), MCL1 (1:3000; Santa Cruz Biotechnology, Paso Robles, CA, USA, sc-819), ATG7 (1:3000; Cell Signaling Technology, 8558 S), LC3B (1:3000; Cell Signaling Technology, 2775 S), EGF (1:1000; Abcam, Cambridge, UK, ab206106), NANOG (1:3000; Bethyl Laboratories, Montgomery, TX, USA, A300-379A), TRPV1 (1:3000; Abcam, ab6166), FLAG (1:5000; Medical & Biological Laboratories, Nagoya, JPN, M185-3L), pAKT (1:3000; Cell Signaling Technology, 9271), AKT1 (1:3000; Cell Signaling Technology, 9272), and β-actin (1:5000; Medical & Biological Laboratories, M177-3) were used. Western blotting was followed by incubation with the appropriate secondary antibodies conjugated to horseradish peroxidase (HRP), anti-rabbit IgG-HRP (1:5000; Enzo, Farmingdale, NY, USA, ADI-SAB-300-J), and anti-mouse IgG-HRP (1:5000; Enzo, ADI-SAB-100-J). The immunoreactive bands were developed with the chemiluminescence ECL Detection System (GE Healthcare, Chicago, IL, USA), and signals were detected using a luminescent image analyzer (LAS-4000 Mini, Fujifilm, JPN).
6
Rabies Virus Infection Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK-293T (human embryonic kidney, 293T), N2a (neuroblastoma), BV2 (mouse microglioma), BSR (cloned from baby hamster kidney-21, BHK-21), and Vero (Cercopithecus aethiops kidney) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher, USA) supplemented with 10%–12% fetal bovine serum (Thermo Fisher). The lab-attenuated RABV strain CVS-B2c (CVS) (derived from CVS-24 virus by passaging in BHK-21 cells), the dog-derived RABV strain DRV-Mexico (DRV) (Yu et al., 2014 (link); Zhang and Fu, 2012 (link)) and the silver-haired bat rabies virus (SHBRV) strain 18 (Morimoto et al., 1996 (link)) was stored in our laboratory. Sendai virus (Cantell strain, Charles River Laboratories) was used at a final concentration of 100 hemagglutinin units per ml. Monoclonal antibodies against FLAG-tag, HA tag, V5-tag, and β-actin were purchased from Medical & Biological Laboratories (MBL, Nagoya, Japan). Monoclonal antibodies (mAbs) against the RABV-N protein RABV-P were prepared in our laboratory. All experiments involving mice were performed following the recommendations in the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of China and were approved by the Scientific Ethics Committee of Huazhong Agricultural University (approval number: HZAUMO-2017-053).
7
Characterization of CADM1 Protein Isoforms
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A mouse L fibroblast subclone that exogenously expressed full-length CADM1 was established previously (Koma et al., 2004 (link)). Rabbit CNT renal distal tubular cells were described previously (Takahashi and Suzuki, 1994 (link)). A recombinant soluble isoform of CADM1 fused with the Fc portion of human IgG1 (sCADM1-Fc) and the deletion form that lost the three Ig-like loops from sCADM1-Fc (ΔsCADM1-Fc) were purified as described previously (Koma et al., 2004 (link); Hagiyama et al., 2009 (link)), and the concentrations were determined using the Bradford assay (Bio-Rad Laboratories, Hercules, CA, United States). Antibodies against the CADM1 ectodomain (3E1 and 9D2, chicken monoclonal) and the C-terminus (rabbit polyclonal) were described previously (Furuno et al., 2005 (link); Hagiyama et al., 2009 (link)). 3E1 and 9D2 were biotinylated using the Biotin Labeling Kit-NH2 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) according to the manufacturer’s instructions. Other primary antibodies used in this study targeted human IgG Fc (goat polyclonal; Jackson ImmunoResearch Laboratories, West Grove, PA, United States) and β-actin (Medical & Biological Laboratories, Nagoya, Japan). Peroxidase-conjugated secondary antibodies were purchased from Amersham (Buckinghamshire, United Kingdom). A chicken IgY clone (U04) was purchased from R&D Systems (Minneapolis, MN, United States).
8
Western Blot Analysis of SV-HUC-1 Cell Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Different groups of SV-HUC-1 cells were harvested, washed with ice-cold phosphate-buffered saline (PBS), and lysed in radioimmunoprecipitation buffer (TNT Bio, #AKR-190, China). A bicinchoninic acid protein assay (Pierce, #23252, Rockford, USA) was used to assess the concentrations of the precipitated proteins in the cell lysates. Then, each sample was diluted to equal concentrations, boiled for 5 min, and separated by 8% SDS-PAGE. Afterwards, the proteins were transferred to PVDF membrane and 5% milk was used to block the membrane at room temperature for 1 h. The membranes were incubated overnight at 4 °C with the following primary antibodies (1:1,000): vimentin (Cell Signaling Technology, #5741), E-cadherin (Cell Signaling Technology, #14472), collagen I (Abcam, #ab138492; 1:500), Slug (Cell Signaling Technology, #9585), Smad2 (Proteintech, #12570-1-AP, USA), phospho-Smad2 (Cell Signaling Technology, #18338, USA); TGF-β (Cell Signaling Technology, #3709, USA); and β-actin (Medical & Biological Laboratories, #M177-3, Japan). Following three washes with PBS + Tween-20, membranes were incubated with the corresponding secondary antibodies for 1 h at room temperature followed by washing and staining. Protein bands were detected using the Immobilon western chemiluminescence HRP substrate kit (Takara, #T7101, China). β-actin served as the loading control.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!