Overexpression of ALS-Linked Proteins

For mammalian expression, SOD1^WT and SOD1^G85R were cloned into the pEF-BOS vector, and TDP-43^WT and TDP-43^Q331K were cloned into the pRK5-Myc vector, as previously described (9 (link), 15 (link)). A Gateway Donor vector encoding human NEDD4L was purchased from the Johns Hopkins Genomic Resources DNA repository (BC032597.1) and recombined in the Destination vector pDEST40 containing a V5 tag. Both pCI-Flag-USP7 and SBE4-Luc plasmids (Addgene 16655 and 16495) were previously described (31 (link)). The Cys223Ser mutation was introduced into the pCI-Flag-USP7 plasmid by site-directed mutagenesis using primer-encoded nucleotide replacements and the HiFi Assembly kit (NEB). The control Flag-GUS plasmid expressing β-glucuronidase was previously described (19 (link)). The HA-ubiquitin–expressing plasmid was purchased from Addgene (18712). The plasmids expressing USP7 shRNAs with a red fluorescent protein (RFP) marker (pRFP-C-RS, catalog no. TF308454) and the scrambled control (catalog no. TR30015) were from OriGene Technologies Inc. The SMAD2 shRNA sequence (5′-CAT​GAT​CCA​GTA​TCA​CAG​TAT-3′) was cloned into an L4R1-H1-promoter plasmid and used with a scrambled shRNA control as described previously (18 (link)).