HFF transfections were performed with the Lonza 4DX Nucleofector using the P2 Primary Cell Solution kit (PBP2-02250) and the NHDF transfection protocol. Transfections were performed with 1–1.5 μg of pEGFP–N1-α-actinin-1 (a gift from Carol Otey, University of North Carolina, Chapel Hill; Addgene plasmid 11908), 1 μg of pCMV-LifeAct-TagRFP (purchased from Ibidi), 1.4 μg of tdTomato–paxillin-22 (a gift from Michael Davidson, National High Magnetic Field Laboratory and Department of Biological Science, Florida State University; deceased 2015; Addgene 58123), pEGFP-MRLC1 (a gift from Tom Egelhoff, Lerner Research Institute, Cleveland Clinic; Addgene 356801), and/or RFP-zyxin (a gift from Anna Huttenlocher, University of Wisconsin–Madison; Addgene plasmid 26720; Bhatt et al., 2002 ).
Rfp zyxin
Manufactured by Addgene
Sourced in Morocco
RFP-zyxin is a fluorescent protein-tagged version of the zyxin protein. Zyxin is a focal adhesion protein involved in cytoskeletal organization and cell-matrix adhesion. The RFP (red fluorescent protein) tag allows for visualization of the zyxin protein within cells.
Automatically generated - may contain errors
Lab products found in correlation
Pcmv lifeact tagrfp, by Ibidi (1 mentions)
4d nucleofector x, by Lonza (1 mentions)
Pegfp n1 α actinin 1, by Addgene (1 mentions)
Nocodazole, by Merck Group (1 mentions)
Latrunculin a, by Merck Group (1 mentions)
Blebbistatin, by Merck Group (1 mentions)
Bovine calf serum (bcs), by Merck Group (1 mentions)
Lifeact mcherry, by Addgene (1 mentions)
Lipofectamin 2000, by Thermo Fisher Scientific (1 mentions)
Y 27632, by Merck Group (1 mentions)
Fugene hd reagent, by Promega (1 mentions)
Lookout mycoplasma pcr detection kit, by Merck Group (1 mentions)
Hek293t cell, by American Type Culture Collection (1 mentions)
3 protocols using rfp zyxin
1
Fluorescent Transfection Techniques for Cell Imaging
2
Cell Imaging with Inhibitor Treatments
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Cells were cultured at 37°C under 5% CO2 with a culture medium, high glucose D-MEM with 1% penstrep (penicillin-streptomycin, Thermo Fisher Scientific) supplemented with 10% bovine calf serum (Sigma). Transfections were performed with Lipofectamin 2000 (Invitrogen) using a standard protocol, and the following constructs were used: mCherry-Lifeact, GFP-NMHC2A (nonmuscle myosin heavy chain 2A, kindly provided by Ewa Paluch lab, UCL), RFP-zyxin (kindly provided by Anna Huttenlocher lab, University of Wisconsin-Madison), or mCherry-MRLC2A (Addgene). For experiments with inhibitors, ROCK inhibitor Y-27632 was used at a concentration of 10 μM, microtubule de-polymerizing agent nocodazole at 10 μM, myosin-II inhibitor blebbistatin at 25 μM, MLCK inhibitor ML-7 at 10 μM, F-actin depolymerizing agent latrunculin A at 1 μM, Arp2/3 inhibitor CK666 at 50 μM (all from Sigma), and lamellipodia growth promoter C8-BPA at 100 μM (Nedeva et al., 2013 (link)). Before drug addition, we performed a control acquisition of at least 1 hr. To prevent flows, defocusing or potential damage of the CDM during manipulation, medium with drugs was added to the running experiment without removing the medium to reach the target concentration.
3
Characterization of MDA-MB-231 Breast Cancer Cells
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MDA-MB-231 (MDA231) human breast cancer cells were provided by P. Siegel (McGill University, Montreal, Canada). MDA231 cells stably expressing an avian Src Y527F mutant (referred to as MDASrc) were obtained from A. Mak (Queen’s University; Hu et al., 2011 (link)), and MDASrc cells were transduced with either EV or ezrin shRNA as will be described. HEK293T cells were obtained from the American Type Culture Collection (Manassas, VA). All cell lines were maintained in DMEM supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich) at 37°C in a humidified atmosphere with 5% CO2. Transient transfections were performed using FugeneHD reagent (Promega, Madison, WI) according to the manufacturer’s protocol. RFP-zyxin was obtained from Addgene (plasmid 26720; Cambridge, MA). GFP-cortactin was provided by A. Craig (Queen’s University). The pCB6 empty vector and WT and T/A ezrin constructs were a kind gift from M. Arpin (Curie Institute, Paris, France). All cell lines were routinely tested for mycoplasma using the Lookout Mycoplasma PCR Detection Kit (Sigma-Aldrich).
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