Cells were transfected with actin (pCAG-mGFP-Actin, (21948) or vimentin (pVimetin-PSmOrange, (31922), (AddGene, Cambridge, MA, USA) plasmids. Cells were seeded at low density (2000–5000 cells/cm2) onto 6-well tissue culture treated plates in antibiotic free medium and allowed to adhere overnight. After this, cells were transfected with plasmids using a specific dermal fibroblast transfection reagent (Cambio, Cambridge, UK). The concentrations of plasmids and reagent were scaled down according to the number of cells per well. The transfection was allowed for 6 hours and after the fresh antibiotic free medium was replaced. All live experiments with transfected cells were performed 48 h after transfection.
Pcag mgfp actin
Manufactured by Addgene
Sourced in United States
The PCAG-mGFP-actin is a plasmid containing a gene that encodes for a fusion protein of mGFP (monomeric Green Fluorescent Protein) and actin. This plasmid can be used to express the mGFP-actin fusion protein in cells.
Automatically generated - may contain errors
Lab products found in correlation
Prccmv vector, by Thermo Fisher Scientific (2 mentions)
Planapon 100x 1.49 oil, by Olympus (2 mentions)
Ix81 system, by Hamamatsu Photonics (2 mentions)
Orca r2 ccd camera, by Hamamatsu Photonics (2 mentions)
Metamorph software, by Molecular Devices (2 mentions)
Cry2phr mcherry, by Addgene (1 mentions)
Pcibn deltanls pmgfp, by Addgene (1 mentions)
7 protocols using pcag mgfp actin
1
Transfection of Fibroblasts with Actin and Vimentin
2
Cloning of Optogenetic Constructs
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Cloning of I-BAR–Cry2–mCh, Cry2–mCh–I-BAR, I-BAR–Cry2–mCh–WH2, and Cry2–mCh–MTSS1 constructs was conducted using a previously described cloning scheme (34 (link)). Briefly, the I-BAR domain from MTSS1, the WH2 domain from MTSS1, and MTSS1 were PCR amplified from human MTSS1 complementary DNA obtained from the Arizona State University DNA repository (DNASU ID: HsCD00746054). N-terminal I-BAR genes were cloned into Cry2PHR–mCherry (Addgene; #26866) using NheI and XhoI restriction sites (23 (link)). C-terminal I-BAR, WH2, and MTSS1 genes were cloned into Cry2PHR–mCherry using BsrgI and NotI restriction sites. The ezrin–GFP expression construct was a gift from Stephen Shaw (plasmid pHJ421; Addgene; #20680 (35 (link))). The actin expression construct (pCAG-mGFP-actin; Addgene; #21948) was a gift from Ryohei Yasuda. CIBN–GFP–CAAX (pCIBN(deltaNLS)-pmGFP; Addgene; #26867) was a gift from Chandra Tucker. Midi prep quantities of DNA of each construct were created from Escherichia coli and collected for cell transfection.
3
Generation of pRcCMV-GFP-Par3 Plasmid
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pRcCMV-GFP-Par3 was generated using pK-myc-Par3 plasmid (gift from I. Macara, Vanderbilt University, Nashville, TN; 58738; Addgene). The myc portion of this plasmid was replaced with the corresponding one of mGFP (amplified from 21948, pCAG-mGFP-actin; Addgene; a gift from R. Yasuda, Duke University, Durham, NC), and the entire insert of the resulting plasmid was then inserted into the pRcCMV vector (Invitrogen) using HindIII/XbaI sites.
4
Live Imaging of Neural Fold Cells
5
Generating GFP-tagged Scribble and Lano Plasmids
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pRcCMV-GFP-Scrib was generated using pKvenus-Scribble plasmid (gift from I. Macara, Vanderbilt University, Nashville, TN; 58738; Addgene). The Venus portion of this plasmid was replaced with the corresponding one of mGFP (amplified from 21948, pCAG-mGFP-actin; Addgene; gift from R. Yasuda, Duke University, Durham, NC) using unique HindIII/BsrGI sites flanking Venus cDNA. The entire insert of the resulting plasmid was then inserted into pRcCMV vector (Invitrogen) using HindIII/XbaI sites. The deletion mutant of hScrib (GFP-sLUR) was produced by PCR, and the resulting PCR product was inserted between BsrGI and XbaI sites of the pRcCMV-mGFP-Scrib. PCR-based mutagenesis of this plasmid was used to generate C-terminal deletion mutants shown in Fig. 8 . To construct pRcCMV-GFP-Lano, the cDNA encoding hLano (RC200125; OriGene) was amplified and inserted between NheI/NotI sites of the pRcCMV-mGFP vector. The eLUR-GFP was constructed in the pRcCMV using a PCR-amplified fragment of the Erbin cDNA encoding the 1–508-aa region. The original plasmid (RG220010) was obtained from OriGene. All plasmid inserts were completely sequenced before use. Some of the plasmids were constructed by DNA Custom Cloning.
6
Imaging and Quantifying Actin Dynamics in Transfected Neurons
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Neurons transfected with morpholinos and pCAG mGFP-Actin (Addgene #21948)
were cultured on the fibronectin-coated glass and imaged after 24 hrs using a PlanApoN 100x/1.49 oil immersion objective on an Olympus IX81 system equipped with a Hamamatsu ORCA-R2 CCD camera. Images were captured every 1 s for up to 3 min. The system was equipped with a focus drift correction mechanism and the imaging was conducted at 37 0 C without CO 2 .
Following generation of substacks of the original movie in Fiji, kymographs were generated using the MetaMorph software (Molecular Devices) using a segmented line tool (width: 5 pixels). Velocities were calculated from the kymographs using the FlowTrack code (obtained from Dr. D. Odde, University of Minnesota; (Chan and Odde 2008) in the Matlab 2007b. Retrogradely moving regions from the heat map generated of the kymograph were selected using a rectangular selection. The analysis was restricted to sections of the time-lapse series where the filopodia were attached and not dynamic.
were cultured on the fibronectin-coated glass and imaged after 24 hrs using a PlanApoN 100x/1.49 oil immersion objective on an Olympus IX81 system equipped with a Hamamatsu ORCA-R2 CCD camera. Images were captured every 1 s for up to 3 min. The system was equipped with a focus drift correction mechanism and the imaging was conducted at 37 0 C without CO 2 .
Following generation of substacks of the original movie in Fiji, kymographs were generated using the MetaMorph software (Molecular Devices) using a segmented line tool (width: 5 pixels). Velocities were calculated from the kymographs using the FlowTrack code (obtained from Dr. D. Odde, University of Minnesota; (Chan and Odde 2008) in the Matlab 2007b. Retrogradely moving regions from the heat map generated of the kymograph were selected using a rectangular selection. The analysis was restricted to sections of the time-lapse series where the filopodia were attached and not dynamic.
7
Imaging and Quantifying Actin Dynamics in Transfected Neurons
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Neurons transfected with morpholinos and pCAG mGFP-Actin (Addgene #21948)
were cultured on the fibronectin-coated glass and imaged after 24 hrs using a PlanApoN 100x/1.49 oil immersion objective on an Olympus IX81 system equipped with a Hamamatsu ORCA-R2 CCD camera. Images were captured every 1 s for up to 3 min. The system was equipped with a focus drift correction mechanism and the imaging was conducted at 37 0 C without CO 2 .
Following generation of substacks of the original movie in Fiji, kymographs were generated using the MetaMorph software (Molecular Devices) using a segmented line tool (width: 5 pixels). Velocities were calculated from the kymographs using the FlowTrack code (obtained from Dr. D. Odde, University of Minnesota; (Chan and Odde 2008) in the Matlab 2007b. Retrogradely moving regions from the heat map generated of the kymograph were selected using a rectangular selection. The analysis was restricted to sections of the time-lapse series where the filopodia were attached and not dynamic.
were cultured on the fibronectin-coated glass and imaged after 24 hrs using a PlanApoN 100x/1.49 oil immersion objective on an Olympus IX81 system equipped with a Hamamatsu ORCA-R2 CCD camera. Images were captured every 1 s for up to 3 min. The system was equipped with a focus drift correction mechanism and the imaging was conducted at 37 0 C without CO 2 .
Following generation of substacks of the original movie in Fiji, kymographs were generated using the MetaMorph software (Molecular Devices) using a segmented line tool (width: 5 pixels). Velocities were calculated from the kymographs using the FlowTrack code (obtained from Dr. D. Odde, University of Minnesota; (Chan and Odde 2008) in the Matlab 2007b. Retrogradely moving regions from the heat map generated of the kymograph were selected using a rectangular selection. The analysis was restricted to sections of the time-lapse series where the filopodia were attached and not dynamic.
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