Human myosin 1e and truncated constructs tagged with EGFP have been previously described78 (link). Human myosin 1f (NM_012335.3) cDNA from the Mammalian Gene Consortium was amplified and cloned into pEGFP-C1 (Clontech). Both myosins were also cloned into mEmerald-C1 and tdTomato-C1 (gifts from Michael Davidson; Addgene plasmids #53975 and 54653) and mScarlet-C1. The following constructs were purchased from Addgene: EGFP-PLCδ-PH, EGFP-AKT-PH, and EGFP-PKCδ-C1 (gifts from Tobias Meyer, plasmids #211789, 21218, and 21216), mScarlet-PM (gift from Dorus Gadella, #98821), EGFP-VAMP3 (a gift from Thierry Galli, #42310), Ruby-Lifeact, mEGFP-Lifeact, mEmerald-Lifeact and EGFP-actin (gifts from Michael Davidson, #54674, #54610, #54148, and #56421). EGFP-FcγRIIA was a gift from Sergio Grinstein (Hospital for Sick Kids, Toronto, ON), and EGFP-TAPP1 was a gift from Aaron Marshall (University of Manitoba, Winnipeg, MB). YFP-myo1c and EGFP-myo1g were gifts from Matt Tyska (Vanderbilt University, Nashville, TN) that were cloned into mEmerald-C1. All primers used for construct generation are listed in Supplementary Table 1 .
Memerald c1
Manufactured by Addgene
The MEmerald-C1 is a fluorescent protein designed for microscopy applications. It emits green fluorescence when excited by blue light. The core function of this product is to serve as a fluorescent tag or marker for visualizing proteins of interest in living cells.
Automatically generated - may contain errors
2 protocols using memerald c1
1
Fluorescent Myosin Constructs in Cells
2
Plasmid Construct Generation for 3D-SIM
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The plasmid used for immortalizing cells, pBabe-puro largeTcDNA (plasmid #14088), and those used to express lamins for 3D-SIM (mEmerald-LaminA-C-18, plasmid #54138; and mEmerald-LaminB1-10, plasmid #54140) were obtained from Addgene (Cambridge, MA). The cDNA fragment encoding LB2 was amplified by PCR from pEGFP-LB2 (Moir et al., 2000 (link)) and inserted into mEmerald-C1 (plasmid #53975; Addgene) using the InFusion HD cloning system (Clontech, Mountain View, CA). The cDNA fragment encoding LC was cut from pEGFP-myc-LC (Shimi et al., 2008 (link)) and inserted into mEmerald-C1 cut with BspEI and BamHI. All DNA constructs were verified by sequencing.
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