Plasmid Construction for Keratin Studies

For in vitro studies, plasmids pT7HMT-K14WT and pT7HMT-K5WT were created by subcloning the relevant cDNAs (1,419 bp for K14WT; 1,773 bp for K5WT) into a pT7HMT modified vector (which features a T7 promoter) using NcoI–BamHI and NdeI–BamHI restriction sites, respectively. For expression in mammalian cells, plasmids pBK-CMV His-GFP-K14WT and pBK-CMV His-GFP-K14C367A were generated by subcloning the cDNAs for K14WT and K14C367A fused to a His-GFP tag at the N terminus into a pBK-CMV vector backbone using SalI–NotI restriction sites (Lee et al., 2012 (link)). The cysteine-free variant of K14 (K14CF) was generated by replacing all five cysteines in K14WT with alanines using a QuikChange lightning multi site-directed mutagenesis kit (Agilent Technologies). Otherwise, cysteine point mutations were introduced into the K14WT cDNA via a QuikChange lightning site-directed mutagenesis kit (Agilent Technologies). For live cell imaging, plasmid pShuttle-CMV-H2B-mCherry was created by subcloning the H2B-mCherry cDNA (1,098 bp) from plasmid PGK-H2BmCherry (Addgene) into a pShuttle-CMV backbone (a gift from B. Vogelstein, Johns Hopkins University School of Medicine, Baltimore, MD) via KpnI–HindIII restriction sites.