Cloning and Expression of Actin-Binding Proteins

Full-length human ACTN1, ACTN4, and ZYX cDNAs were obtained by polymerase chain reaction (PCR) using DLD-1 cell cDNA as a template. The following deletion mutants of ZYX were amplified by PCR: ZYX-N (amino acids [aa] 1–51) and ZYX-C (aa 52–572). All cDNAs were sequenced and then subcloned into the pCMV2A, pCMV4A (Agilent Technologies, Santa Clara, CA, USA), pEGFP-N2, pEGFP-N3, pEGFP-C1, pmCherry-N1 (TAKARA BIO Inc., Otsu, Japan), pGEX-6P-1 (GE Healthcare, Piscataway, NJ, USA), and pFastBacHT A (Life Technologies). The GFP-vinculin (VCL) construct was previously described [35 (link)]. For ACTN stable expression in DLD-1 cells, cDNAs encoding GFP, ACTN1-GFP, and ACTN4-GFP were amplified by PCR and inserted into pIRESpuro3 vector (TAKARA BIO Inc.).
Plasmids were transfected in DLD-1 or SW480 cells using Lipofectamine 2000 reagent or in FreeStyle 293F cells with FreeStyle Max reagent according to the manufacturer’s instructions (Life Technologies).