Pcmv6 ac gfp mammalian expression vector

mCldn15 complementary DNA cloned in the pCMV6-AC-GFP mammalian expression vector was purchased from Origene (Rockville, MD, USA), hCldn14 cDNA was purchased from Genecopoeia (Rockville, MD), and the mCldn14 cDNA was derived from Nunes et al.^{32 (link)}. Site-directed mutagenesis was performed using the KAPA HiFi PCR kit (Kapa Biosystems, Inc., Wilmington, MA, USA) or the Agilent Quickchange Lighting kit (Agilent, Santa Clara, CA). All cell lines used in this study were originally obtained from American Type Culture Collection. COS7 or HEK293T cells were seeded in 35 or 60 mm cell culture plates and cultured in Dulbecco’s modified Eagle medium (Invitrogen) supplemented with 10% fetal bovine serum. Cells were transfected with plasmids encoding for WT or mutant Cldn using Lipofectamine LTX (Life Technologies, Carlsbad, CA) and the cells were allowed to express the protein for at least 24 h. Each transfection and subsequent analysis was carried out at least twice.

Endotoxin‐free plasmid DNA purification. DH5α competent cells (Invitrogen) were transformed by heat‐shock as previously described [9 (link), 10 (link)] using DFFB Human Tagged ORF Clone (Origene, RG237731) or pCMV6‐AC‐GFP Mammalian Expression Vector (Origene, PS100010).
Plasmid DNA purification was performed using Nucleobond^® Xtra Maxi EF kit (Macherey‐Nagel) and plasmid concentration using Nucleobond^® Finalizer (Macherey‐Nagel).
Transfections. Cell‐line‐specific Nucleofector Kits (Lonza) used were and transfections were performed in accordance to manufacturers protocols. Nucleofection efficiency was determined by RT‐qPCR 24 hours post transfection.