Ub r gfp

Plasmids for the overexpression of SGTA-V5 and Pex19-V5 were previously described [43 (link)]. Ub-M–GFP (#11938) and Ub-R–GFP (#11939) plasmids described in [45 (link)] were purchased from Addgene. HA tagged USP5 (#22590) was purchased from Addgene and subcloned into pcDNA5 mammalian expression vector. The plasmid for the expression and USP5^C335A mutant was created by site directed mutagenesis using the HA tagged USP5 as template and primers carrying the mutated codons. DNA sequencing was used to verify the mutant prior to use. The 3x NNP/AAA (NNP positions at 226–228, 239–241 and 255–257) variant of SGTA-V5 was previously described [46 (link)].
The pET28-TxA-SGTA wild type plasmid for the expression and purification of WT SGTA was previously described [46 (link)]. This plasmid was generated by cloning WT SGTA into BamHI/XhoI restriction sites of a pET28c vector modified to encode an N-terminal thioredoxin A (TxA) fusion protein followed by a hexa-histidine tag. Plasmids for the expression and purification of SGTA_TPR double mutant (SGTA^K160E/R164E) and SGTA_UBL double mutant (SGTA^D27R/E30R) mutant were created by site directed mutagenesis using pET28-TxA-SGTA wild type plasmid as template and primers carrying the mutated codons. DNA sequencing was used to verify the mutants prior to use.

HeLa (5 × 10⁵) cells were seeded in 6-well culture plates. Next day, cells were transfected with Ub^G76V-GFP (Addgene plasmid # 11941), Ub^R-GFP (Addgene plasmid # 11939), and Ub^M-GFP (Addgene plasmid # 11938) reporter constructs (valuable gifts from Nico Dantuma) [37 (link)] by using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA) as the manufacturer recommended. Next day cells were treated with vehicle (0.1% v/v Et-OH) for 24 h or OTA (10 µM) alone or in combination with proteasome inhibitors, VR23 (5 µM) and Epoxomicin (250 nM). After 24 h, GFP signal was visualized with inverted fluorescence microscope (Eclipse TS100, Nikon, Tokyo, Japan) and quantitatively analyzed by flow cytometry using C6 Acuri, BD (Becton Dickinson, Franklin Lakes, NJ, USA).

Ub-G76V-GFP and Ub-R-GFP were purchased from addgene. Cells were transfected with plasmids using lipofectamine 3000 (invitrogen L3000008) according to the manufacturer’s protocol.