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Ptre gfp nmhc 2 a

Manufactured by Addgene

PTRE-GFP-NMHC II-A is a plasmid construct that expresses green fluorescent protein (GFP) under the control of a tetracycline-responsive promoter (PTRE). The plasmid also contains the non-muscle myosin heavy chain II-A (NMHC II-A) gene sequence.

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3 protocols using ptre gfp nmhc 2 a

1

Generating HA-tagged α-catenin Mutant Constructs

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Expression vectors containing the HA-tagged α-catenin mutant were previously described (Ozawa, 1998 (link); Matsubara and Ozawa, 2001 (link)). A CAG vector containing HA-tagged α-catenin (pC-αHA) (Taniguchi et al., 2005 (link)) was digested with NotI and EcoRV, and the fragment containing full-length α-catenin cDNA was cloned into the NotI/EcoRV site of pU-DNCT (Ozawa and Kobayashi, 2014 (link)), a derivative of pUHD10-3 (Gossen and Bujard, 1992 (link)), yielding pTRE-α-cateninFLAG. pCAGGShyg, which confers hygromycin resistance, and the pCAG/bsr-7 vector, which confers blasticidin resistance gene, were described previously (Ozawa and Kobayashi, 2014 (link); Ozawa, 2015 (link)). pZeoSV2(+), which contains the zeocin-resistance gene, and the gRNA expression vector containing the α-1,3-galactosyltransferase gene were provided by Masahiro Sato (Kagoshima University). pTRE-GFP–NMHCIIA (Addgene plasmid # 10844) were gifts from Robert Adelstein (Wei and Adelstein, 2000 (link)).
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2

Transfection of Myh9 and Myh10 Plasmids

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Expression plasmids that encode human Myh9, CMV-GFP-NMHC II-A (Addgene plasmid 11,347) or pTRE-GFP-NMHC II-A (Addgene plasmid 10,844), and its control isoform Myh10, NMHC II-B (Addgene plasmid 11,348 and Addgene plasmid 10,845), were obtained from Dr. Robert Adelstein through Addgene, a nonprofit plasmid repository (http://www.addgene.org/) [35 (link),36 (link)]. Expression plasmids were transfected into RAW 264.7 cells using Lipofectamine® LTX Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s recommendations. In some cases, gene expression was induced by addition of Doxycycline (1 μg/ml) for the expression plasmids utilizing the Tet-on system (pTRE-GFP-NMHC II-A).
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3

Cloning NM2 Heavy Chains into Fluorescent Vectors

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The NM2 heavy chains were cloned into pEGFP-C3 with a TET promoter (pTRE-GFP-NMHC II-A, Addgene ID: #10844, pTRE-GFP-NMHC II-B, Addgene ID: #10845)53 (link). The NM2 heavy chain contructs were kindly provided by Robert S. Adelstein’s lab (National Heart, Lung and Blood Institute, Bethesda, MD, USA).
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