On target plus human smart pool

VPS26A was subcloned from an original plasmid, which was a gift from R. Teasdale (University of Queensland, St. Lucia, Australia; Kerr et al., 2005 (link)), into the lentiviral vector pXLG3. To produce siRNA-resistant VPS26A six silent base mismatches (C187T, A189G, A192G, C195T, A198G, and A201G) were introduced into the ORF, generating resistance to VPS26A siRNA oligonucleotide 1 from the ON-TARGET plus human SMART pool (GE Healthcare). To produce siRNA-resistant VPS26B seven silent base mismatches (G795A, C798T, T799A, C800G, G804A, C805A, and C807G) were introduced into the ORF, generating resistance to VPS26B siRNA oligonucleotide 2 from the ON-TARGET plus human SMART pool (GE Healthcare; Gallon et al., 2014 (link)). The VPS26A (p.K93E), VPS26A (p.M112V), and VPS26A (p.K297X) mutations were generated using site-directed mutagenesis using the following primers: VPS26A (p.K93E): 5′-GTTCTCCAGGTAAGGCTAGTTCCTCCACTAGGTTTACAAATTCATGA-3′; VPS26A (p.M112V): 5′-GACTCAGAGCAGAAGTTATGATTTTGAATTTGTGCAAGTTGAAAAGCC-3′; VPS26A (p.K297X): 5′-GAAGTCCGGAGTTCACTAAGCATATTAGAAGCATGTTTTAG-3′.

RPE-1 cells stably expressing GFP, wild-type or mutant VPS26A were transfected either with a ON-TARGET plus nontargeting control pool (GE Healthcare; sequences: 5′-UGGUUUACAUGUCGACUAA-3′, 5′-UGGUUUACAUGUUGUGUGA-3′, 5′-UGGUUUACAUGUUUUCUGA-3′, and 5′-UGGUUUACAUGUUUUCCUA-3′) or with the VPS26A suppression oligonucleotide 1 (sequence 5′-GCUAGAACACCAAGGAAUU[DTDT]-3′) and VPS26B suppression oligonucleotide 2 (sequence 5′-GAAGUUCUCUGUGCGCUAU[DTDT]-3′) of the ON-TARGET plus human SMART pool (GE Healthcare) to suppress endogenous VPS26A and VPS26B. Cells were reverse-transfected using DharmaFECT (GE Healthcare), then transfected again 12 h later according to manufacturer’s instructions. 48 h after the second transfection, cells were fixed and stained.