Usp14

pcDNA3-PD-L1 and pcDNA3-HA-PD-L1 have been described previously^{17 (link)}. Flag-USP8 (WT and mutants: amino acid 1–313, 314–714, 715–1118, and C786A) and Flag-TRAF6 (WT and mutants: amino acid 1–259, 1–348, 260–499, and C70A) were amplified and cloned into pcDNA3-Flag vector. USP8 and TRAF6 were amplified and cloned into pLenti-HA vector. HA-tagged TRAF1, TRAF2, TRAF3, and Skp2 have been described previously^{50 (link)}. Flag-TRAF2, TRAF3, TRAF4, TRAF5, TRAF7, USP2, USP7, USP10, USP13, USP14, and USP20, TRAF2 were purchased from Origene. PD-L1-Luciferase constructs and E3 ligase library for luciferase screening were provided by the Laboratory of Dr. Hong-Bing Shu. His-Ub and mutants have been described previously^{51 (link)}. shRNAs targeting USP8 were purchased from Open Biosystems. shRNA sequences for mouse Usp8: 5ʹ-TTGTAAGCATTAGATGTGAGG-3ʹ(#744); 5ʹ-TAGCATTGGTTGTAAACTGCG-3ʹ(#745). shRNA sequences for mouse p65: 5ʹ-ATGGATTCATTACAGCTTAAT-3ʹ(#1); 5ʹ-CGGATTGAGGAGAAACGTAAA-3ʹ(#2). sgRNAs for human TRAF6: 5ʹ-GTAACAAAAGATGATAGTGT-3ʹ(#5); 5ʹ-TGGGTGGAACTGCCAGCACG-3ʹ(#6). sgRNA for human USP8 was 5ʹ-GATTTTACTTATCCCTCATTGG-3ʹ. sgRNAs for mouse Usp8: 5ʹ-TGAAGAAAAGGACAGACGGG-3ʹ(#1); 5ʹ-GGTCTTTTAGTGAAGAACTG-3ʹ(#2). sgRNAs for mouse Traf6: 5ʹ-CCTCTCCAGCTCCTTCATGG-3ʹ(#4); 5ʹ-GCAGTATTTCATTGTCAACT-3ʹ(#5). Control sgRNA sequence: 5ʹ-CTTGTTGCGTATACGAGACT-3ʹ(#1); 5ʹ-CGCTTCCGCGGCCCGTTCAA-3ʹ(#2).

Plasmids containing myc-TDP-43 and myc-TDP-43 M337V were generated as described previously [30 (link)]. Plasmids for tau (4R/2N), α-synuclein, USP14 and UCHL5 were purchased from Origene. Mutant plasmids USP14 C114A, USP14 WT ΔUBL, USP14 C114A ΔUBL and UCHL5 C88A were generated from the wildtype plasmid by Genscript. Cells were treated with IU1 (25–100 μM; Selleckchem or Sigma), MG132 (10 μM; Selleckchem), PS341 (10 μM; Selleckchem) and b-AP15 (0.5–2 μM; Selleckchem or Millipore). All compounds were dissolved in DMSO (Sigma), and control samples were treated with the same volume of DMSO.