Primary neonatal rat CFs were isolated from hearts of 2- to 3-day-old SD rats as previously described [14 (link)]. Cells were cultured in DMEM containing 1% penicillin and streptomycin and 10% fetal bovine serum (FBS) in a 5% CO2 atmosphere at 37 °C. Primary cultured CFs were identified by primary antibodies against α-smooth muscle actin (α-SMA; Cell Signaling Technology, MA, USA) and vimentin (Proteintech, IL, USA) (Fig. S2 ). CFs from passages 3 to 5 were replaced with the serum-free medium for 24 h when the cell density reaches 70%, and treated with the absence and presence of apelin-13 (100 nM; TOCRIS, Bristol, UK), miR-122 inhibitor (50 nM; GenePharma, Shanghai), miRNA negative control (50 nM), ELA (100 nM; MedChemExpress, NJ, USA), recombinant human ACE2 (rhACE2, 0.1 ng/µl; MedChemExpress, NJ, USA), and N-acetyl-L -cysteine (NAC, 5 mM; Sigma-Aldrich, MO, USA), respectively. The abovementioned agents were added to CFs for 30 min prior to 24h exposure of Ang II (1 μM; Sigma-Aldrich, MO, USA). The treated cells were then subjected to in vitro experiments. The primary CMs were treated with Ang II for 24 h and then subjected to quantitative reverse transcription PCR analysis.
α smooth muscle actin α sma
Manufactured by Cell Signaling Technology
Sourced in United States
α-smooth muscle actin (α-SMA) is a protein that is a component of the cytoskeleton in smooth muscle cells. It plays a role in the contraction and regulation of smooth muscle tissue.
Automatically generated - may contain errors
Lab products found in correlation
E cadherin, by Cell Signaling Technology (4 mentions)
Pvdf membrane, by Merck Group (3 mentions)
N cadherin, by Cell Signaling Technology (3 mentions)
Vimentin, by Cell Signaling Technology (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Phospho smad2 ser465 ser467, by Cell Signaling Technology (2 mentions)
Smad2, by Cell Signaling Technology (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
N acetyl l cysteine nac, by Merck Group (1 mentions)
Vimentin, by Proteintech (1 mentions)
Apelin 13, by Bio-Techne (1 mentions)
Mir 122 inhibitor, by GenePharma (1 mentions)
Ang 2, by Merck Group (1 mentions)
Annexin 5 fitc and pi staining kit, by BD (1 mentions)
Sodium bicarbonate, by Merck Group (1 mentions)
Tmrm assay kit, by Thermo Fisher Scientific (1 mentions)
Gapdh 60004 1 ig, by Proteintech (1 mentions)
Cell counting kit 8 (cck8), by Apexbio (1 mentions)
Molecular imager chemidoc xrs system, by Bio-Rad (1 mentions)
20 protocols using α smooth muscle actin α sma
1
Isolation and culture of primary neonatal rat cardiac fibroblasts
2
Mefunidone Induced Kidney Injury Amelioration
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Mefunidone (Lot No. 21062601) was synthesized by the Pharmaceutical Sciences Department, Central South University (Changsha, China). Folic acid (FA) (F8758) and sodium bicarbonate (V900182) were purchased from Sigma-Aldrich. Antibodies against proteins NGAL (ab216462) and collagen Ⅰ (ab270993) were purchased from Abcam. Antibodies against Bax (#2772), cleaved-caspase3 (#9664), and α-smooth muscle actin (α-SMA) (#19245) were purchased from Cell Signaling Technology. Antibodies against proteins bcl2 (#T40056) were purchased from Abcam. Antibodies against OxPhos (45–8,099) were purchased from Invitrogen. Antibodies against E-cadherin (20874-1-AP), vimentin (60330-1-Ig), α-tubulin (66031-1-Ig), and GAPDH (#60004-1-Ig) were purchased from Proteintech. A tissue reactive oxygen species assay kit (DHE) (#D7008) was purchased from Sigma-Aldrich. FITC Annexin V and PI staining kit (556547) was purchased from BD Pharmingen. Cell counting kit-8 (K1018) was purchased from APExBIO Technology LLC. The TMRM assay kit (T668) was purchased from Thermo Fisher Scientific. Blood urea nitrogen (BUN) (C013-2-1) and serum creatinine (SCr) (C011-2-1) kits were obtained from the Nanjing Jiancheng Bioengineering Institute. All the chemicals used in this study were of analytical grade.
3
Western Blot Analysis of EMT Markers
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RIPA buffer (Beyotime, China) added protease inhibitors was employed to extract total proteins from cells. Concentration of total proteins was determined by a BCA kit (Thermo Scientific, USA). Equivalent proteins were subjected to SDS-polyacrylamide gels and electotransferred onto PVDF membranes (Millipore, USA). The membranes were the incubated with primary antibodies and secondary antibodies in turn. Protein bands were visualized at a Molecular Imager ChemiDoc XRS System (Bio-Rad, USA). The following primary antibodies were used: Smad2 (#5339, Cell Signaling Technology, USA); Phospho-Smad2 (Ser465/Ser467) (#18338, Cell Signaling Technology, USA); Smad3 (#9523, Cell Signaling Technology, USA); Phospho-Smad3 (Ser423/425) (#9520, Cell Signaling Technology, USA); α-Smooth Muscle Actin (α-SMA) (#19245, Cell Signaling Technology); N-cadherin (#13116, Cell Signaling Technology); E-cadherin (ab1416, Abcam); CCND1 (ab16663, Abcam); p21 (ab188224, Abcam); Connective tissue growth factor (CTGF) (ab6992, Abcam); Zinc Finger E-Box Binding Homeobox 1 (ZEB1) (ab181451, Abcam); GAPDH (ab9485, Abcam).
4
Histological Analysis of Kidney Damage
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After the rats had been sacrificed, the ligated kidneys were fixed in 4% paraformaldehyde for histological examination. Kidney tissue was embedded in paraffin and cut into 3-μm-thick sections. Standard sequential techniques were used to dewax the sections before haematoxylin-eosin (HE) and Masson’s trichrome (MT) staining. Damage was assessed as described in previous studies [11 (link), 12 (link)].
The kidney sections used for immunohistochemical detection were incubated with primary antibodies against α-smooth muscle actin (α-SMA) (1:1000, Cell Signaling Technology, Inc., USA) and E-cadherin (1:1000, Cell Signaling Technology, Inc., USA) according to the manufacturer’s protocols. After washing, the sections were further incubated with anti-rabbit secondary antibody (Abcam, Cambridge, USA) at a 1:5000 dilution. Finally, the sections were counterstained with Mayer’s haematoxylin and dehydrated for further observation. The Graphic context analysis software ImagePro plus 6.0 was used to analyse the immunohistochemistry pictures. Five fields were chosen under a × 200 microscope (OLYMPUS U-LH100-3) for each slice to record the positive staining for the average integral optical density.
The kidney sections used for immunohistochemical detection were incubated with primary antibodies against α-smooth muscle actin (α-SMA) (1:1000, Cell Signaling Technology, Inc., USA) and E-cadherin (1:1000, Cell Signaling Technology, Inc., USA) according to the manufacturer’s protocols. After washing, the sections were further incubated with anti-rabbit secondary antibody (Abcam, Cambridge, USA) at a 1:5000 dilution. Finally, the sections were counterstained with Mayer’s haematoxylin and dehydrated for further observation. The Graphic context analysis software ImagePro plus 6.0 was used to analyse the immunohistochemistry pictures. Five fields were chosen under a × 200 microscope (OLYMPUS U-LH100-3) for each slice to record the positive staining for the average integral optical density.
5
Epithelial-Mesenchymal Transition in Lung Cancer
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Human lung adenocarcinoma A549 and NCI-H1299 cell was phurchased from Shanghai Institute of Biochemistry and Cell Biology. Antibodies against CTEN, TGF-β1, E-cadherin, N-cadherin, Vimentin, α-smoothmuscle actin (α-SMA) and decapentaplegic homolog 2 (Smad2) were purchased from Cell Signaling Technology Inc. p-decapentaplegic homolog 2(p-Smad2) was purchased from Abcam Inc. Lipofectamine 2000, Trizol and reverse-transcription kit were purchased Invitrogen Inc. Transwell chamber and matrigel were purchased from BD Inc. Puromycin, ECL chemiluminescence staining solution, and PVDF membrane were phuchased from Sigma Inc. RPMI-1640 medium was phuchased from Gibco Inc.
6
Cell Signaling Pathway Inhibitors Assay
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Anisomycin, SB203580 [45 (link)], SP600125, and ferrostatin-1 [46 (link)] were purchased from MCE (Shanghai, China) and dissolved in dimethyl sulfoxide (DMSO). Cells were treated with anisomycin at the indicated concentration or coincubated with SB203580 (20 μM) or ferrostatin-1 (4 μM). Commercially available antibodies were used in this study. The anti-tubulin antibody was obtained from InTech (Shanghai, China), while anti-c-Myc (#18583), CD133 (#64326), Nanog (#4903), EpCAM (#2929), caspase-3 (#14220), Bcl-2 (#15071), Bax (#5023), N-cadherin (#13116), E-cadherin (#3195), vimentin (#5741), α-smooth muscle actin (α-SMA, #19245), p38 MAPK (#8690), phospho-p38 MAPK (p-p38 MAPK, #9216), histone H3 (#4499), phospho-histone H3 (Ser10) (p-H3S10, #53348), phospho-histone H3 (Ser28) (p-H3S28, #9713), NRF2 (#12721), SLC7A11 (#12691), FTH1 (#4393), and NCOA4 (#66849) antibodies were purchased from Cell Signaling Technology (Boston, USA). Anti-CD24 (ab31622) antibody was provided by Abcam (Cambridge, UK).
7
Cultured Aortic SMCs Drug Treatments
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Rat or mouse aortic SMCs were isolated and grown in DMEM supplemented with 10% fetal bovine serum and antibiotics. SMCs were starved with serum‐free DMEM for 24 hours before drug treatments. BW723C86, LY272015, SP600125, SB202190, PD98059, LY294002, and U73122 were from Tocris Bioscience (Bristol, UK). 5‐HT, platelet‐derived growth factor‐BB (PDGF‐BB), 5‐bromo‐2′‐deoxyuridine (BrdU), and Pluronic F‐127 powder were from Sigma‐Aldrich (St. Louis, MO). Rapamycin was from Cell Signaling Technology (Beverly, MA). Antibodies against mammalian target of Rapamycin (mTOR), phosphorylated mTOR (Ser2448), p70S6 kinase, phosphorylated p70S6 kinase (Ser389), α‐smooth muscle actin (α‐SMA), and SM22α were from Cell Signaling Technology. Antibody against 5‐HT2BR was from BD Bioscience Pharmingen (San Diego, CA). Antibodies against 5‐HT receptor 2A (5‐HT2AR) and vimentin were from Abcam (Cambridge, MA). Antibodies against BrdU and CD31 were from Santa Cruz Biotechnology (Dallas, TX). Antibody against Mac‐2 was from Celdarlane (Burlington, ON, Canada).
8
Western Blot Profiling of EMT Markers
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Cells (HepG2, Hep3B, and Huh7 cells) and liver tissues were lysed using the EzRIPA Lysis kit (ATTO Corporation, Tokyo, Japan) and quantified using Bradford reagent (Bio-Rad). Proteins were visualized by Western blot analysis using primary antibodies (see below; 1:1000 dilution) at 4°C overnight and then with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000 dilution) for 1 h at 25°C. The primary antibodies included the antibodies against E-cadherin, N-cadherin, Snail, vimentin, sirtuin 1 (SIRT1), matrix metalloproteinase-2 (MMP-2), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), α-smooth muscle actin (α- SMA), and β-actin, all of which were obtained from Cell Signaling Technology (Danvers, MA). Specific immune complexes were detected using the Western Blotting Plus Chemiluminescence Reagent (Millipore, Bedford, MA).
9
Atrial Tissue Protein Analysis
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Right atrial (RA) and left atrial (LA) tissue homogenates were probed with the following antibodies: gp91phox (BD Transduction); total and phosphorylated RyR2 at serine-2808 and serine-2814 (ThermoFisher Scientific); α-smooth muscle actin (α-SMA; Cell Signalling); vimentin (Exbio); HRP-conjugated GAPDH (Sigma Aldrich); and total CaMKII (Cell Signalling) and an antiserum to oxidized CaMKII that was kindly provided by Dr ME Anderson, Johns Hopkins University, USA. Detection of primary antibodies was performed using peroxidase-conjugated anti-rabbit or anti-mouse antibodies (both Promega, USA). GAPDH was used as a loading control except for phosphorylated protein levels which were normalized by the respective total protein levels. All western blots were run under reducing conditions apart from those to detect oxidized CaMKII, which was performed under non-reducing conditions to preserve the redox modification. For the detection of RA and LA NOX2, atrial tissues were pooled from two different animals prior to homogenization.
10
Mycobacterium tuberculosis Stimulation Pathway
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Heat-killed Mycobacterium tuberculosis H37Ra (MTBRa) (Difco Lab, Detroit, MI, USA) and ET-1 (R & D System; Minneapolis, MN, USA) were dissolved in phosphate-buffered saline (PBS) and used as a stimulant [18 (link)]. The antibodies to α-smooth muscle actin (α-SMA) and E-cadherin were purchased from Cell Signaling Technology (Beverly, MA, USA), and those to ET-1, collagen I, fibronectin were obtained from R & D System (Minneapolis, MN, USA), Santa Cruz (Dallas, TX, USA), Novus Biologicals (Littleton, CO, USA), respectively. The antibodies to mesothelin, AKT, IκBα and α-tubulin were from Thermo Fisher Scientific (Waltham, MA, USA). The ET receptor antagonists BQ123, BQ788 and Bosentan were acquired from Sigma (St. Louis, MO, USA). Mitogen-activated protein kinase kinase (MEK) inhibitor PD98059, c-Jun N-terminal kinase (JNK) inhibitor SP600125, p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580, nuclear factor (NF)-κB inhibitor parthenolide and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 were obtained from Calbiochem (San Diego, CA, USA).
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