Establishing Stable Cell Lines with GFP-Rab19 and GFP-MYH9

The pLVX-GFP-Rab19 lentiviral transfer plasmid used to establish the GFP–Rab19 stable cell lines was previously described (Jewett et al., 2021 (link)). A pLVX-GFP-MYH9 transfer plasmid was cloned by ligating a NdeI/SalI-digested GFP-MYH9 cassette from CMV-GFP-NMHC II-A (Addgene plasmid #11347, deposited by Robert Adelstein) into NdeI/XhoI-digested pLVX-Puro vector. For lentiviral vector production, the transfer plasmid was co-transfected with the packaging plasmid pΔ8.9 and pseudotyping plasmid pVSV-G into HEK293T cells.
The pGEX-KG-Rab19 plasmid, encoding GST-tagged Rab19 for expression in E. coli for recombinant protein production, was previously described (Jewett et al., 2021 (link)). The HA-Vps3 expression plasmid was a gift from Dr Jacques Neefjes (Cell & Chemical Biology, LUMC, The Netherlands), and the Vps8–GFP expression plasmid was a gift from Dr Judith Klumperman (Cell Biology, UMC Utrecht, The Netherlands).