Plasmids were propagated in NEB 5-alpha competent E. coli cells (New Englad Biolabs) and purified with QIAprep spin miniprep kits (Qiagen) or the PureYield plasmid maxiprep system (Promega) before transfection.
Constructs for human myosin Va (amino acids 1–1091), myosin VI (amino acids 1–1021), calmodulin, and FHL3-Halo were reported previously1 (link). Human Zyxin-Halo constructs were subcloned into a pCAG mammalian expression vector with a C-terminal 3C protease cleavage site and eGFP tag for affinity purification. Human VASP-Halo constructs were subcloned into a pET15b bacterial expression vector containing an N-terminal Strep-Tag II and 3C cleavage site, as well as a C-terminal 3C site and 6xHis-Tag. Human ɑ-actinin-Halo constructs were subcloned into a pET15b bacterial expression vector containing an N-terminal 6xHis-Tag and Tobacco Etch Virus (TEV) cleavage site. Human profilin in a pMW plasmid was a generous gift from Henry Higgs, Dartmouth University. Human Zyxin-mNeonGreen and F-tractin-mScarlet were subcloned into a pM98 mammalian expression vector. The template for mScarlet was a gift from Dorus Gadella (University of Amsterdam) via Addgene (plasmid #85054). All plasmids were assembled with Gibson cloning, and point mutations were generated by site-directed mutagenesis.
Constructs for human myosin Va (amino acids 1–1091), myosin VI (amino acids 1–1021), calmodulin, and FHL3-Halo were reported previously1 (link). Human Zyxin-Halo constructs were subcloned into a pCAG mammalian expression vector with a C-terminal 3C protease cleavage site and eGFP tag for affinity purification. Human VASP-Halo constructs were subcloned into a pET15b bacterial expression vector containing an N-terminal Strep-Tag II and 3C cleavage site, as well as a C-terminal 3C site and 6xHis-Tag. Human ɑ-actinin-Halo constructs were subcloned into a pET15b bacterial expression vector containing an N-terminal 6xHis-Tag and Tobacco Etch Virus (TEV) cleavage site. Human profilin in a pMW plasmid was a generous gift from Henry Higgs, Dartmouth University. Human Zyxin-mNeonGreen and F-tractin-mScarlet were subcloned into a pM98 mammalian expression vector. The template for mScarlet was a gift from Dorus Gadella (University of Amsterdam) via Addgene (plasmid #85054). All plasmids were assembled with Gibson cloning, and point mutations were generated by site-directed mutagenesis.
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