Journal: Cell reports

Article Title: TRIM21-dependent target protein ubiquitination mediates cell-free Trim-Away.

doi: 10.1016/j.celrep.2023.112125

Figure Lengend Snippet: Figure 6. Complex heterotypic K48/K63 ubiquitin chains are assembled on Trim-Away targets Ubiquitin chain restriction (UbiCRest) analysis of biotinylated HpaII after cell- free Trim-Away (see experimental setup in Figure S5A). The target protein was immobilized on streptavidin-coupled magnetic beads prior to Trim-Away in HSS containing proteasome inhibitor (MG-262). After Trim-Away for 20 min, the beads were washed and subjected to deubiquitinase (DUB) treatment, comparing non-specific USP2, K48 linkage-specific OTUB1*, K63-specific AMSH*, or both OTUB1* and AMSH*. Digested samples were analyzed by SDS-PAGE and western blotting, visualizing biotinylated HpaII (A), total ubiquitin (B), K63-linked ubiquitin chains (C), or K48-linked ubiquitin chains (D). In lanes 10 and 11, K48- and K63-linked ubiquitin chains assembled in vitro were separated alongside to confirm the specificities of ubiquitin antibodies and for comparing released polyubiquitin species after DUB treatment. The low-molecular-weight sections in (B)–(D) were exposed longer than the high- molecular-weight regions to support the identification of unattached ubiquitin species. Asterisks correspond to cross-reacting bands. See also Figure S5.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Lysozyme from chicken egg white Sigma Cat# L6876 Glutathione Sepharose 4B Cytiva Cat# GE17-0756-01 AminoLinkTM Coupling Resin Thermo Fisher Scientific Cat# 20382 DynabeadsTM Protein A Invitrogen Cat# 10001D BSA Fisher Scientific Cat# BP1600-100 Nocodazole Sigma Cat# M1404 ATP Sigma Cat# A5394 Phosphocreatine Sigma Cat# P6502 Creatine Phosphokinase Sigma Cat# C3755 MLN7243 Selleck Chemicals Cat# S8341 MG-262 R&D Systems Cat# I120200 NMS-873 Sigma Cat# SML1128 MLN4924 Active Biochem Cat# A-1139 Recombinant Human USP2 Catalytic Domain Protein, CF R&D Systems Cat# E-506 DynabeadsTM M-280 Streptavidin Invitrogen Cat# 11206D Proteinase K Roche Cat# 3115879001 [a-32P]dATP Perkin Elmer Cat# BLU512H500UC Recombinant Human Ubiquitin Protein, CF R&D Systems Cat# U-100H Recombinant Human Ubiquitin Activating Enzyme (UBE1), CF R&D Systems Cat# E-305 Recombinant Human His6-UBE2W Isoform 1 Protein, CF R&D Systems Cat# E2-740 Recombinant Human His6-UBE2N (Ubc13)/Uev1a Complex, CF R&D Systems Cat# E2-664 Recombinant Human UbcH5b/UBE2D2 Protein, CF R&D Systems Cat# E2-622 Apyrase New England Biolabs Cat# M0398L Critical commercial assays Reductive Alkylation Kit Hampton Research Cat# HR2-434 TnT SP6 High-Yield Wheat Germ Protein Expression System Promega Cat# L3260 Experimental models: Organisms/strains Xenopus laevis (females) Nasco Cat# LM0053MX Xenopus laevis (males) Nasco Cat# LM00715MX Recombinant DNA pOPINK Ray Owens Addgene Cat# 41143 HLTV-hTRIM21 Clift et al.9 Addgene Cat# 104973 pTM192_pOPINK_hTRIM21R-R-PS This paper N/A pTM193_pOPINK_UbWT-hTRIM21R-R-PS This paper N/A pTM216_pOPINK_h TRIM21R-R-PS_K251only (referred to as DK) This paper N/A pTM231_pOPINK_UbK63R-hTRIM21R-R-PS This paper N/A pTM38_pOPINK_xlTFIIS This paper N/A pTM209_pOPINK_xlTFIIS_DK This paper N/A pOPINB-AMSH* Michel et al.39 Addgene Cat# 66712 pOPINB-OTUB1* Michel et al.39 Addgene Cat# 65441 16 Cell Reports 42, 112125, February 28, 2023

Techniques: Ubiquitin Proteomics, Magnetic Beads, SDS Page, Western Blot, In Vitro, Molecular Weight, High Molecular Weight