Flag-tagged PKP2 has been described previously (Chen et al., 2002 (link)). Full-length human small interfering RNA (siRNA)-resistant PKP2.FLAG (p1376) has been described previously (Bass-Zubek et al., 2008 (link)). To generate enhanced green fluorescent protein (EGFP)-tagged PKP2 (p1381) and mCherry-tagged PKP2 (p1383) in LZRS for epithelial cell transduction human PKP2 was amplified from a cDNA library and cloned first into pCMV5a.FLAG (p915). PKP2 was subsequently amplified from p915 and subcloned into pEGFP-C1 to generate p964. Retroviral vectors LZRS-EGFP and LZRS-mCherry were generated by removing the fluorescent tags from pEGFP-C1 and pmCherry-C1 and ligating them into LZRS-pBMN (p989). The human PKP2 from p964 was ligated into LZRS-pEGFP to generate p1381 and into LZRS-pmCherry to generate p1383. Human actin-pmCherry (p1246), a gift from Shin-ichiro Kojima (Gakushuin University, Tokyo Japan) and Gary Borisy (Marine Biological Laboratory, Woods Hole, MA) was ligated into an LZRS-shuttle vector to generate p1263, followed by ligation into LZRS to generate p1309. The DP.GFP construct and the inducibly expressing cells used in the live cell imaging experiments have been described previously (Godsel et al., 2005 (link)). Ecad.red fluorescent protein (RFP) was a gift from W. J. Nelson (Stanford University, Palo Alto, CA) and used to construct LZRS-Ecad-EGFP (p1400) by subcloning the Ecad sequence into pEGFP-N1 followed by ligation of the Ecad-EGFP insert into the LZRS-pBMN retroviral vector. For Ecad-mCherry (p1401) the EGFP tag from p1400 was replaced by the mCherry tag from the pmCherry vector.
Construction of the glutathione S-transferase (GST)-tagged Rhotekin Rho-binding domain (GST-RBD) prokaryotic expression construct was described in Dubash et al. (2007) (link), and this construct, the nucleotide-free GST-RhoA mutant (G17A), and the EGFP-RBD plasmid were the gifts of K. Burridge (University of North Carolina at Chapel Hill). siRNA against human PKP2, against human p120ctn and nontargeting siRNAs were used for knockdown (KD) experiments (Thermo Fisher Scientific, Waltham, MA).
Construction of the glutathione S-transferase (GST)-tagged Rhotekin Rho-binding domain (GST-RBD) prokaryotic expression construct was described in Dubash et al. (2007) (link), and this construct, the nucleotide-free GST-RhoA mutant (G17A), and the EGFP-RBD plasmid were the gifts of K. Burridge (University of North Carolina at Chapel Hill). siRNA against human PKP2, against human p120ctn and nontargeting siRNAs were used for knockdown (KD) experiments (Thermo Fisher Scientific, Waltham, MA).
