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5 protocols using emtb 3xgfp

1

Live Imaging of Early Crozier Embryos

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We synthesized mRNAs for microinjections with Ambion’s SP6 mMESSAGEmMACHINE kit. The capped mRNAs produced were diluted in nuclease-free water and used for microinjections in order to detect fluorescence signal in early M. crozieri embryos. Nuclei were marked and followed using histone H2A-mCherry (H2A-mCh) and GFP-Histone (H2B-GFP). The plasmids carrying the nuclear marker pCS2-H2B-GFP (GFP-Histone) and pDestTol2pA2-H2A-mCherry [37 (link)] were transformed, purified and concentrated as described before and then linearized with the restriction enzymes NotI and BglII, respectively. To follow live microtubules, we used a GFP fusion of the microtubule binding domain of ensconsin (EMTB-3XGFP). These clones were the gift of the Bement Lab (University of Wisconsin) [8 (link), 56 (link)] and were commercially ordered from http://addgene.org (EMTB-3XGFP: https://www.addgene.org/26741/).
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2

Fluorescent Protein Transfection Workflow

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Cells were grown overnight in 24-well plates at 37°C in a 5% CO2 atmosphere. After reaching over 80% confluence, the plasmids mRuby-Clathrin (Addgene #55852), pEGFP-Sec23A (Addgene #66609), ZsGreen-Rab5 (custom synthesized by Genomeditech (Shanghai, China) Co., Ltd.), EGFP-Rab7A (Addgene #28047), GFP-LAMP1 (Addgene #16290), EMTB-3XGFP (Addgene #26741), pSNAPf-Cox8A (Addgene #101129), or pSNAPf-TOMM20 (custom synthesized by Genomeditech (Shanghai, China) Co., Ltd.) was transfected into the cells using Lipofectamine 3000 (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. After 24 h, the transfected cells were digested with trypsin-EDTA and seeded into Nunc Glass Bottom Dishes (Φ 12 mm, Thermo Fisher Scientific, Inc.) at a density of 1.5 ~ 2.0 × 104 per well in growth medium (150 μL). The cells were grown for an additional 12 ~ 24 h before incubation with the indicated probes.
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3

Plasmid Amplification and Mutagenesis

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Plasmid amplification was performed via transformation in E. coli (XL1-Blue) and DNA isolation via MIDI-prep (NucleoBond® Xtra Midi, Macherey & Nagel, #740410). The respective amber stop mutants were generated by introducing a TAG codon through PCR-based site-directed mutagenesis. The plasmid for expression of the TNF-receptor pTNFR1-tdEOS was a gift from Mike Heilemann (Addgene plasmid #98273). The plasmid for the expression of the Kainate receptor (pcDNA3-GluK2) was a gift from Dr. Peter Seeburg (Max-Planck Institute for Medical Research, Heidelberg, Germany). An AgeI restriction site was introduced at the C-terminus of the GluK2 coding sequence in front of the STOP-codon. Subsequently, GluK2 was cloned into the MCS of the pEGFP-N1 plasmid (Clontech #U55762) using the restriction enzymes EcoRI and AgeI. The plasmid for expression of the microtubule-associated protein Ensconsin (EMTB-3xGFP) was a gift from William Bement (Addgene plasmid #26741). The plasmid for the expression of the tRNA/tRNA-synthetase pair (pCMV tRNAPyl/NESPylRSAF) was kindly provided by Edward Lemke53 (link).
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4

Diverse Fluorescent Protein Constructs

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mcherry, eGFP, eGFP-α-tubulin, eGFP-βactin, and eGFP-Mito were obtained from Clontech. eGFP-VASP [Svitkina et al. 2003 (link)] was a gift from the Borisy lab. eGFP-Utrophin [Burkel et al. 2007 (link)] was a gift from the Bement lab. eGFP-Lifeact [Riedl et al. 2008 (link)] and eGFP-F-tractin [Johnson and Schell 2009 (link)] were gifts from the Baum lab. EMTB-3xGFP [Miller and Bement 2009 (link)] was a gift from William Bement (Addgene plasmid #26741). PM-GFP [Teruel et al. 1999 (link)] was a gift from Tobias Meyer (Addgene plasmid # 21213). EGFP-H2B-6 (Addgene plasmid # 56436), mcherry-LaminA-C-18 (Addgene plasmid # 55068), and mcherry-Vimentin-7 (Addgene plasmid # 55156) [Shaner et al. 2007 (link)] were a gift from Michael Davidson. pcDNA3-HtrA2-EGFP [Martins et al. 2002 (link)] was a gift from L. Miguel Martins (Addgene plasmid # 14121). For simplicity, all text references referring to “GFP” are describing enhanced-GFP constructs.
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5

Fluorescent protein-tagged cell imaging

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Cells were grown overnight in 24-well plates at 37°C in a 5% CO 2 atmosphere. After reaching over 80% confluence, the plasmids mRuby-Clathrin (Addgene #55852), pEGFP-Sec23A (Addgene #66609), ZsGreen-Rab5 (custom synthesized by Genomeditech (Shanghai, China) Co., Ltd.), EGFP-Rab7A (Addgene #28047), GFP-LAMP1 (Addgene #16290), EMTB-3XGFP (Addgene #26741), pSNAPf-Cox8A (Addgene #101129), or pSNAPf-TOMM20 (custom synthesized by Genomeditech (Shanghai, China) Co., Ltd.) was transfected into the cells using Lipofectamine 3000 (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. After 24 h, the transfected cells were digested with trypsin-EDTA and seeded into Nunc Glass Bottom Dishes (Φ 12 mm, Thermo Fisher Scientific, Inc.) at a density of 1.5~2.0 × 104 per well in growth medium (150 µL). The cells were grown for an additional 12~24 h before incubation with the indicated probes.
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