Cmv gfp nmhc 2 a

Human myo1e and myo1f constructs tagged with EGFP have been previously described (Barger et al., 2019 (link)). mEmerald-Lifeact was a gift from Michael Davidson (Addgene #54148). Cofilin-EGFP was a gift from James Bamburg (Addgene #50859). Chicken regulatory light chain (RLC) tagged with EGFP was a gift from Klaus Hahn. WASP tagged with myc was a gift from Dianne Cox, and was subcloned into a pUB-Halo-C1 vector. CMV-GFP-NMHCII-A was a gift from Robert Adelstein (Addgene #11347). ARP3-mCherry (Addgene #27682) and mCherry-cortactin (Addgene #27676) were gifts from Christien Merrifield that were subcloned into EGFP-C1 and mEmerald-C1, respectively. Chicken paxillin was a gift from Chris Turner, which was subcloned into mScarlet-i-C1. Chicken vinculin was a gift from Kenneth Yamada (Addgene #50513) and subcloned into pUB-mEmerald-C1. Immunohistochemical staining to determine localization of select podosome-related proteins in relation to the actin teeth did not produce good results, which may be due to the adhesive and porous nature of the IgG-functionalized DAAMPs. As an alternative, we transfected the RAW macrophages with fluorescently tagged proteins. All transfections were accomplished by electroporation (Neon) using the manufacturer’s instructions.

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).

For stable transfections, HN30 and HN31 cells were cultured in 6-well plates until they reached 70-80% confluency. The cells were transfected with CMV-GFP-NMHC II-A (Addgene plasmid # 11347) using 6 μg of using NanoJuice Transfection Reagent in serum-free medium (Novagen) according to the manufacturer's protocol [19 (link)]. HN30 and HN31 cells were cultured for 7-14 days in 400 μg/ml of geneticin before being sorted for selection of stable clones.