Ha ub k48

HA-Ub (WT), HA-Ub (K6) only, HA-Ub (K11) only, HA-Ub (K27) only, HA-Ub (K29) only, HA-Ub (K33) only, HA-Ub (K48) only, HA-Ub (K63) only, and HA-GASC1 were purchased from Addgene. HA-Ub (K63R) was generated by cloning the corresponding cDNAs into the pCMV-HA vector via XbaI/NotI sites. pCMV-Myc-ROCK2, pCMV-Myc-ROCK2-CAT (aa 5-553), and pCMV-Myc-ROCK2-RB/PH (aa 686-1388) were generated by subcloning the corresponding cDNAs into the pCMV-Myc vector via KpnI/XhoI or XbaI/NotI sites. HA-FBXO42 and HA-FBXO42-△F-box (deleting aa 44-93) was generated by subcloning the corresponding cDNAs into the pCMV-HA vector via XhoI/BamHI sites. pCMV-Myc-ROCK2-K121R was generated using the Quick Change Q5 Site-Directed Mutagenesis Kit (NEBaseChanger) according to the manufacturer’s instructions. The pLKO.1-puro lentiviral MISSION shRNA constructs targeting endogenous GASC1 (shGASC1 CDS1 (TRCN0000022056): sense, 5′-GCCTCTGACATGCGATTTGAA-3′, and shGASC1 CDS2 (TRCN0000022055): sense, 5′-CCTTGCATACATGGAGTCTAA-3′), FBXO42 (shFBXO42 CDS (TRCN0000134822): sense, 5′-CCATCAGTGTTATCATGGTTT-3′, and a non-targeting (NT) control shRNA (TRC1/1.5) were from Sigma-Aldrich. Details of plasmid construction are available upon request.

The mammalian expression plasmids Flag-tagged TBK1 and V5-tagged SRA were stored in our laboratory. Flag-CYLD, Flag-TBK1 KD, ULD, SDD, CTD, 30R, 401R, and 30R/401R were obtained from BGI (BGI group, Shenzhen, China). HA-Ub, HA-Ub-K48, HA-Ub-K63, and HA-Ub-K33 were purchased from Addgene (http://www.addgene.org). The HEK293T cells were cultured in DMEM high glucose supplemented with 10% FBS. Cells were seeded in 10-cm dishes and were transfected with 10 μg plasmid with PEI (Polysciences, Warrington, PA, USA) according to the manufacturer’s instruction.

Flag-TRAF4 (#RC200345) and Flag-MCL1 (#RC200521) were obtained from OriGene Technologies, Inc. His-Ub (#31815), HA-Akt (#73408), HA-Ub-K48 (#17605), and Flag-JOSD1 (#22547) were obtained from Addgene (Watertown, MA, USA). Flag-TRAF4 (C18A), HA-Ub-K48R, His-Ub (K6, K11, K27, K29, K33, K48, and K63), HA-K8/K14R, Flag-MCL1 (S159D), and Flag-MCL1 (S159A) mutant were obtained using the Q5 Site-Directed Mutagenesis Kit (cat. #E0554S; New England BioLabs, Inc.) according to the manufacturer’s instructions. All mutant constructs were generated using mutagenic PCR and verified by Sanger DNA sequencing.