Introduction of Abcc6 Mutations via USER Cloning

Mutagenesis was performed as described previously [19 (link)]. Briefly, mutations were introduced into the Gateway entry vector pEntr223-rAbcc6 by uracil-specific excision reagent (USER) cloning with the primers listed in Table 2 using Phusion U PCR master mix (Thermo Scientific, Waltham, MA, USA). PCR fragments were purified using the Nucleospin gel and PCR cleanup kit (Macherey-Nagel, Düren, Germany) and assembled using the USER enzyme mix (New England Biolabs, Ipswich, MA, USA), according to the manufacturer’s instructions. Resulting circular constructs were verified by Sanger sequencing and transformed into competent E. coli DH5alpha cells. The cDNAs encoding pEnter223-rAbcc6 mutants were subsequently subcloned into a Gateway compatible pQCXIP expression vector using LR Clonase-II (Thermo Scientific, Waltham, MA, USA).