Ez capture mg imaging system

A total of 10–50 μg protein was extracted from cells and used for Western blot analysis. Briefly, the cells were lysed using protein lysis buffer (50 mM Tris–Cl, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 200 mM Na3VO4, 1x proteinase inhibitor, pH 7.4) and the protein concentration was measured using a Micro BCA^TM Protein Assay kit (Thermo Scientific), based on the standard curve of BSA. The antibodies used for immunoblot analysis were purchased and assessed for equal loading as follows: rabbit anti-USP7 (#A300-034, dilution ratio 1:1000) and rabbit anti-USP47 (#A301-048A, 1:1000) from Bethyl Laboratories; mouse anti-Flag (#F1804, 1:1000) from Sigma Aldrich; rabbit anti-ubiquitin (#3933, 1:2000) from Cell Signaling; mouse anti-p53 (#sc-126, 1:1000), and mouse anti-HSP90α/β (#sc-13119, 1:5000) from Santa Cruz Biotechnology; rabbit anti-β-actin (#LF-PA0207, 1:5000) from AbFrontier. Samples were analyzed using SDS-PAGE, and chemiluminescence was measured using the Ez-Capture MG imaging system (ATTO Corporation).

After harvest, cells were lysed using protein lysis buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 50 mM NaF, 5 mM β-glycerophosphate, 1 mM Na₃VO₄, protease inhibitor). Western blot analysis was performed using 10–50 μg protein extracts from cells, and protein concentration was measured by using a Micro BCA^TM protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) with a standard curve using BSA. Antibodies that were used for Western blot analysis were as follows: rabbit anti-USP14 (A300-920A, Bethyl Laboratories, Montgomery, TX, USA, 1:2000), rabbit anti-FASN (#3180S, Cell Signaling Technology, Danvers, MA, USA, 1:1000), mouse anti-HA (sc-7392, Santa Cruz Biotechnology, Dallas, TX, USA, 1:1000), mouse anti-Flag (F1804-1MG, Sigma-Aldrich, 1:1000), and rabbit anti-His (#2365S, Cell Signaling Technology, 1:1000). Rabbit anti-β-actin (LF-PA020, AbFrontier, Seoul, Korea, 1:5000) and mouse anti-HSP90α/β (sc12119, Santa Cruz Biotechnology, 1:5000) were used for loading control. An Ez-Capture MG imaging system (ATTO Corporation, Amherst, NY, USA) was used to detect each protein.