Ubg76v gfp

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).

To obtain reporter plasmid 3xPSMA4-ARE-Luc, three copies of the ARE sequence (TGACTCTGCA) from the promoter region of the human PSMA4 gene were inserted into the pGL4.37 Vector (Promega, Madison, WI, USA) using XhoI and SacI restriction enzymes, as previously described [5 (link)]. Co-reporter plasmid encoding renilla luciferase pRL-TK (E2241) was purchased from Promega (Madison, WI, USA). Constructs pCMV-dR8.2 (Addgene, #8455) and pCMV-VSV-G (Addgene, #8454) required for lentiviral transduction were a kind gift from Bob Weinberg (MIT, Boston, MA, USA). Plasmid Ub^G76V-GFP (Addgene, #11941) was a kind gift from Nico Dantuma, and construct pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-GFP (Addgene, #71237) was a kind gift from Charles Gersbach.

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.