Anti usp2

Spautin-1 (S7888), Imatinib (S1026), and MG132 (S2619) were from Sellectchem (Houston, TX, USA). SKP2-C25 (M60136) was from Xcessbio Biosciences, Inc. (San Diego, CA). MTS reagent (G3582) was from Promega Corporation (Madison, WI, USA). Co-IP assay kit (14311D) was from Life Technologies (Carlsbad, CA). Antibodies: anti-Ubiquitin (#3936), anti-K63-Ubiquitin (#12930), anti-K48-Ubiquitin (#12805), anti-USP10 (#8501), anti-USP1 (#8033), anti-USP2 (#8036), anti-USP7 (#4833), anti-USP8 (#11832), anti-USP14 (#11931), anti-USP15 (#66310), anti-USP18 (#4813), anti-UCHL1 (#13179), anti-UCHL3 (#8141), anti-CYLD (#8462), anti-A20 (#5630), anti-SKP2 (#2652), anti-p27 (#3686), anti-FLAG (#14793), anti-HA (#3724), anti-phospho-c-Abl(Y245) (#2861), anti-c-Abl (#2862), anti-phospho-STAT5 (#9359), anti-STAT5 (#25656), anti-phospho-Crkl (#3181) and anti-Crkl (#38710) (Cell Signaling Technology, Beverly, MA, USA); anti-UCHL5 (ab124931), anti-USP13 (ab109264) (Abcam, Cambridge, MA); anti-GAPDH (BS60630) (Bioworld Technology, Inc., Louis Park, MN, USA).

Western blotting was performed on various cancer cell lines to investigate the alteration of protein expression as described previously [35 (link),36 (link)]. The harvested cells were lysed using RIPA lysis buffer and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The protein was transferred onto nitrocellulose membrane (GE Healthcare Life Science, Pittsburgh, PO, USA) and were incubated with primary antibodies overnight, and then the secondary antibody was incubated at room temperature for 2 h. Finally, expression of proteins was detected by an enhanced chemiluminescence kit (Merck Millipore, Darmstadt, Germany). The information on primary antibodies was provided as below: anti-Bcl-2 and anti-DR4 from Abcam (Waltham, MA, USA); anti-Mcl-1 and anti-cIAP2 from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-Bax, anti-Bim, and anti-XIAP from Biosciences (San Jose, CA, USA); anti-survivin from R&D System; anti-Bcl-xL, anti-cIAP1, anti-DR5, anti-PARP, anti-USP2, and anti-cleaved caspase-3 from Cell Signaling Technology (Beverly, MA, USA); anti-c-FLIP and anti-caspase3 from Enzo Life Sciences (San Diego, CA, USA). RT-PCR and quantitative PCR were used to analyze mRNA expression, and primer sequences were described previously [37 (link)].