Hbx 41108

The following antibodies were used in this study: mouse anti-Flag mAb (used 1:1000 in WB, F1804), rabbit anti-GAPDH mAb (used 1:10000 in WB) (G9545), mouse anti-α-tubulin mAb (used 1:10000 in WB, 05–829) and mouse anti-vinculin (used 1:500 in WB, V9131) from Sigma-Aldrich (St. Louis, MO, USA); mouse anti-DLC1 mAb (used 1:500 in WB and IF, 612021) and mouse anti-FAK mAb (used 1:1000 in WB, 610088) from BD Biosciences (Heidelberg, Germany); rabbit anti-paxillin pAb (used 1:500 in WB and IF, sc-5574) and mouse anti-His mAb (used 1:200 in WB, sc-8036) from Santa Cruz Biotechnology (Dallas, TX, USA); rabbit anti-USP7 mAb (WB 1:1000, 4833), rabbit anti-GFP mAb (WB 1:1000, 2956) and rabbit anti-HA mAb (WB 1:1000, 3724) were from Cell Signaling (Danvers, MA, USA); rabbit anti-HECTD1 pAb (WB 1:1000, 20605–1-AP) was from Proteintech (Manchester, UK). HRP-labeled secondary anti-mouse and anti-rabbit IgG antibodies were purchased from and Dianova (Hamburg, Germany), Alexa-Fluor®-labeled secondary IgG antibodies were from Invitrogen (Carlsbad, CA, United States). Inhibitors used were: Bortezomib from UBPBio (Dallas, TX), MG-132 from Selleck Chemicals (Houston, TX); PR-619, HBX 41108 and P5091 from Cayman Chemicals (Ann Arbor, MI); cycloheximide from Santa Cruz (Dallas, TX).

Flag-USP13, Flag-USP28, HA-CYLD lysine 63 deubiquitinase (CYLD), HA-USP46, pRetroSuper, pRetro-shUSP28-1, and pRetro-shUSP28-3 plasmids were purchased from Addgene (Watertown, MA, USA). HA-PIM1 and HA-PIM2 were gifts from Dr. Andrew Kraft. USP28-Myc, GFP-PIM3, and Flag-F-box and WD repeat domain-containing 7 (FBW7) were created using Gateway cloning. Cells were cultured to approximately 70% confluency, at which point plasmids were transfected using Lipofectamine 3000 (Thermo Fisher, Waltham, MA, USA).
MG-132, PR-619, and AZD5383 were purchased from Selleck Chemical (Houston, TX, USA). HBX 41108 was purchased from Cayman Chemical (Ann Arbor, MI, USA). Cycloheximide (CHX) was purchased from VWR (Randor, PA, USA).