Subcloning, transfection, and stable cell lines generation

XX Xiao Xiang
SL Stéphanie Langlois
MS Marie-Eve St-Pierre
AB Anna Blinder
PC Philippe Charron
TG Tyson E. Graber
SF Stephanie L. Fowler
SB Stephen D. Baird
SB Steffany A. L. Bennett
TA Tommy Alain
KC Kyle N. Cowan
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pcDNA3.1-MCS-Bir*A(R118G)-HA was a gift from Kyle Roux (plasmid #36047, Addgene, Cambridge, MA) [31]. PANX1 cDNA (Origene, Rockville, MD) was subcloned into pcDNA3.1-MCS-BirA*(R118G)-HA. PANX1-BirA*(R118G)-HA, and Myc-PANX1 cDNA (Origene) were subcloned into the pCDH-CuO-MCS-EF1-GFP lentiviral vector (System Biosciences, CA). All constructs were verified by sequencing. See “Supplementary Material and methods” for detailed information.

Transfections were performed using Lipofectamine 2000 Reagent (Thermo Scientific, Waltham, MA). SparQTM Cumate Switch Inducible System (System Bioscience) was used to generate stable cell lines [10]. Cumate (System Bioscience) was used at 30 µg/mL.

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