Qiafilter plasmid maxi prep kit

pcDNA3.1 vectors (Invitrogen, Breda, the Netherlands) containing either mouse sFlt-1-VSV or the luciferase gene, both of which are driven by the cytomegalovirus promoter, were constructed as described previously [19 (link)]. The plasmids were amplified in Escherichia coli DH5α (Invitrogen), purified using the QIAfilter Plasmid Maxi-prep kit (Qiagen, Venlo, the Netherlands) and dissolved in EndoFree Tris–EDTA buffer (Qiagen). The mice were co-transfected with the sFlt-1-VSV and luciferase constructs in both calf muscles (20 μg each) using electroporation, as described previously [19 (link)]. To monitor transfection efficiency, the mice were injected with i.p. luciferin at 2-week intervals. Five minutes after the luciferin injection, luciferase activity was visualised using a NightOWL bioluminescence camera (Xenogen Ivis Spectrum, Alameda, CA, USA), as described previously [19 (link)].

pcDNA4 plasmids encoding wt ERp29 or ERp29 C157S [32 (link)] were used in these experiments. Plasmid maxi-preps were prepared using the QIAfilter Plasmid Maxi-Prep kit (Qiagen). Transient transfections of Min-6 were performed with Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s protocol. Assays were performed 72 hours after transfection as described below.

The transfection protocol was described previously with one adjustment (40 μg sFlt1-VSV DNA instead of 20 μg per muscle) [6 (link),29 (link)]. In brief, two pcDNA3.1 vectors (Invitrogen, Breda, the Netherlands) were constructed, containing either mouse sFlt1-VSV or the luciferase gene, driven by the same cytomegalovirus promoter. The plasmids were amplified in Escherichia coli DH5α (Invitrogen), purified using the QIAfilter Plasmid Maxi-prep kit (Qiagen, Venlo, The Netherlands) and dissolved in EndoFree Tris-EDTA buffer (Qiagen). The mice were co-transfected by electroporation with the sFlt1-VSV and luciferase constructs (40:1) in both gastrocnemius muscles (40 μg DNA each). To monitor transfection efficiency, the mice were anesthetized and injected intraperitoneally with D-luciferin (150 mg/kg; Synchem OHG, Altenburg, Germany) weekly. Five minutes after the luciferin injection, luciferase activity was visualized using a NightOWL bioluminescence camera (Xenogen Ivis Spectrum, Alameda, CA, USA).