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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Lab products found in correlation

Lipofectamine ltx reagent, by Thermo Fisher Scientific (1 mentions) Cmv gfp nmhc 2 a, by Addgene (1 mentions)

3 protocols using ptre gfp nmhc 2 a

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)).

, & Ozawa M. (2018). Nonmuscle myosin IIA is involved in recruitment of apical junction components through activation of α-catenin. Biology Open, 7(5), bio031369.

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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).

Kanzaki H., Movila A., Kayal R., Napimoga M.H., Egashira K., Dewhirst F., Sasaki H., Howait M., Al-dharrab A., Mira A., Han X., Taubman M.A., Nichols F.C, & Kawai T. (2017). Phosphoglycerol dihydroceramide, a distinctive ceramide produced by Porphyromonas gingivalis, promotes RANKL-induced osteoclastogenesis by acting on non-muscle myosin II-A (Myh9), an osteoclast cell fusion regulatory factor. Biochimica et biophysica acta, 1862(5), 452-462.

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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).

Horváth Á.I., Gyimesi M., Várkuti B.H., Képiró M., Szegvári G., Lőrincz I., Hegyi G., Kovács M, & Málnási-Csizmadia A. (2020). Effect of allosteric inhibition of non-muscle myosin 2 on its intracellular diffusion. Scientific Reports, 10, 13341.

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