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4 protocols using antibodies for vimentin

1

Immunoblotting for Cytoskeletal Proteins

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Cell lysate preparation, immunoblotting, and protein quantification were done as previously described [8 (link)]. The antibody against α-SMA (1:1,000) was from Dako. The antibodies for vimentin (1:1,000) and N-cadherin (1:1,000) were from Cell Signaling. The secondary antibodies were fluorescently-conjugated IRDye 680 and IRDye 800 from LI-COR Biosciences (1:20,000). Proteins on the membrane were visualized and quantified by the Odyssey infrared imaging system (LI-COR Biosciences).
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2

Isolation and Characterization of Cardiac Cell Types

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Dulbecco's modified Eagle's medium (DMEM)/F12 (1:1) and foetal bovine serum were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Trypsin, type II collagenase and 5-bromodeoxyuridine were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). DH5α competent cells were purchased from Tiangen (Beijing, China). Specific rabbit monoclonal antibodies against hyperpolarization-activated cyclic nucleotide-gated cation channel 4 (HCN4), which is a marker for SAN, connexin 43 (COX43), which is a common connexion between cardiac cells, cardiac troponin I (cTnI), which is a marker for NRVMs, α-striated actin (α-SA), which is a marker for NRVMs, and GAPDH were purchased from Abcam (Cambridge, MA, USA); antibodies for vimentin, which is a marker for CFs, and COX-45, which is another common connexion between cardiac cells, were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA) and Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), respectively; and antibodies for myosin heavy chain (MHC), which is a marker for NRVMs, and α-striated muscle actin (α-SMA), which is a marker for cardiac myofibroblasts (CMFs) were purchased from Wuhan Sanying Biotechnology (Wuhan, China) and Wuhan Tiandeyue Biotechnology (Wuhan, China), respectively.
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3

Antibodies for Epithelial-Mesenchymal Markers

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Antibodies for Vimentin (1:2000; #5741, Cell Signaling Technology), E-cadherin (1:1000; #14472, Cell Signaling Technology), AXIN2 (1:2000; #5863, Cell Signaling Technology), MYC (1:5000; #13987, Cell Signaling Technology), β-catenin (1:1000; #8480, Cell Signaling Technology), phospho-β-catenin (T41, S45) (1:1000; #9565, Cell Signaling Technology), GSK3β (1:1000; #12456, Cell Signaling Technology) and GAPDH (1:5000; #5014, Cell Signaling Technology) were bought from Cell Signaling Technology. α-catenin (1:2000; #610194, BD) was from BD Transduction Laboratories. Fibronectin (1:1000; SAB4500974, Sigma) and LGR5 (1:1000; SAB2700211, Sigma) were from Sigma.
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4

Western Blot Analysis of Proteins

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Antibodies against GAPDH were from Santa Cruz Biotechnology, (Santa Cruz, CA). Antibodies for vimentin were from Cell Signaling Technology Inc. (Beverley, MA). HRP-conjugated secondary antibodies were from eBioScience (San Diego, CA). Human recommend TGF-β1 was obtained from R&D system (Minneapolis, MN, USA).
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