Psilv u6

Four shRNAs of Nischarin (Nis-shRNA1-4), which target four common sequences of mouse and rat Nischarin (5’-CACAACTGTCGCAACCGC-3’, 5’-TGATGCCAAGACTGACCTT-3’, 5’-CCTCAGAGACAACCGGATT-3’ and 5’-AGCATTGCCGAGGTTGAAA-3’), and the corresponding scrambled shRNAs were synthesized by GeneCopoeia (Guangzhou, China) and subcloned into the lentiviral vector psiLv-U6 (GeneCopoeia, Guangzhou, China), which contains an eGFP coding sequence. To generate lentiviral particles, psiLv-U6 shRNAs were transfected into 293T Lentiviral packaging cellswith a Lenti-Pac™ FIV packing mix and an EndoFectin Lenti transfection reagent. Viral supernatants were harvested, concentrated, aliquoted, and stored at −80°C until use. Titers of the lentiviral stocks were assessed using 10-fold serial dilutions to transfect HEK293T cells together with an eGFP reporter to identify the infected cells.

The DNA plasmid pIRES-BKCa was a gift from Dr. J. D. Lippiat (Dept. of Cell Physiology and Pharmacology, University of Leicester, Leicester, UK) [12 (link)]. The vector plasmid was used as negative control. Transfection was performed using Lipofectamine 2000 and Opti-MEM Reduced-Serum medium (Invitrogen, Carlsbad, CA, USA) as described previously. Cells were transfected with plasmid for 48 h before functional assays were carried out. BKCa downregulation was achieved using the retroviral expression vector psiLv-U6 (GeneCopoeia, Rockville, MD, USA). The shRNA sequence against BKCa was 5′-GTGGGTCTGTCCTTCCCTACT-3′. Viruses were generated and used to infect target cells. Stable clones were selected using puromycin.