Memerald lifeact

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.

Human myosin 1e and truncated constructs tagged with EGFP have been previously described^{78 (link)}. Human myosin 1f (NM_012335.3) cDNA from the Mammalian Gene Consortium was amplified and cloned into pEGFP-C1 (Clontech). Both myosins were also cloned into mEmerald-C1 and tdTomato-C1 (gifts from Michael Davidson; Addgene plasmids #53975 and 54653) and mScarlet-C1. The following constructs were purchased from Addgene: EGFP-PLCδ-PH, EGFP-AKT-PH, and EGFP-PKCδ-C1 (gifts from Tobias Meyer, plasmids #211789, 21218, and 21216), mScarlet-PM (gift from Dorus Gadella, #98821), EGFP-VAMP3 (a gift from Thierry Galli, #42310), Ruby-Lifeact, mEGFP-Lifeact, mEmerald-Lifeact and EGFP-actin (gifts from Michael Davidson, #54674, #54610, #54148, and #56421). EGFP-FcγRIIA was a gift from Sergio Grinstein (Hospital for Sick Kids, Toronto, ON), and EGFP-TAPP1 was a gift from Aaron Marshall (University of Manitoba, Winnipeg, MB). YFP-myo1c and EGFP-myo1g were gifts from Matt Tyska (Vanderbilt University, Nashville, TN) that were cloned into mEmerald-C1. All primers used for construct generation are listed in Supplementary Table 1.