Xl10 gold ultracompetent cells

Mutant plasmids were generated with QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent, Santa Clara, CA, USA) using vector pR-M10-αGalA [12 (link)] as wild type template and following the manufacturer’s instructions. Specific primers (Supplementary Table S1) were used to introduce selected mutations, which affect protein folding (p.Asp170Val, p.Pro205Ser, p.Gln279Arg, p.Arg301Gln), in GLA.
PCR products were used to transform XL10-Gold ultracompetent cells (Stratagene, (part of Thermofisher, Fair Lawn, NJ, USA) and mutated plasmids were purified using the DNA extraction kit NZYMiniprep (Nzytech, Lisboa, Portugal) before sequencing by the Sanger method to confirm the presence of the introduced mutations.
Finally, mutated plasmid maxipreps were prepared from transformed E. Coli, using the HiSpeed Plasmid Maxi Kit (Qiagen, Hilden, Germany).

The coding sequence of LRP6 (LRP6-WT) (NM_002336) is cloned into the 4.7 kb vector PCMV6XL4 (PcM), which encodes ampicillin resistance and is suitable for mammalian cell over-expression assays. The PcM and LRP6-WT plasmids were purchased from OriGene^® Technologies. Mutated plasmids were generated separately by site-directed mutagenesis using a Agilent^® Technologies QuickChange II XL Site-Directed Mutagenesis Kit, according to manufacturer’s instruction. Briefly, the mutant strand was synthesized by a thermal cycling reaction using a high-fidelity DNA polymerase and complementary mutagenic primers. This reaction was followed by a DpnI digestion of the parental methylated and hemimethylated DNA. The DNA vector containing the desired variant was then amplified into XL10-Gold^® Ultracompetent Cells by ThermoFisher^® Scientific and extracted using the NucleoBond^® Xtra Midi Plus kit by Machery-Nagel. The constructs were verified by Sanger sequencing with primers spanning the coding sequence of LRP6.