Ab155993

The experiments were performed according to the previous report [9 (link)]. Proteins were quantified using a BCA protein assay kit (Thermo Scientific, Waltham, MA), and 25 μg proteins were separated using 10% SDS-PAGE and transferred to a PVDF membrane (Millipore). The antibodies for USP5 (1 : 500, #ab155993), TRAF6 (1 : 500, #ab33915), p65 (1 : 500, #ab16502), phos-p65 (1 : 500, #ab76302), IκBα (1 : 500, #ab32518), phos-IκBα (1 : 500, #ab133462), β-actin (1 : 1000, #ab179467), Myc (1: 1000, #ab32), Flag (1 : 1000, #ab49763), and K48-ub (1 : 500, #ab140601) were all bought from Abcam (Abcam, Cambridge, USA).

Western blot analysis was performed using standard techniques. Briefly, pancreatic cancer cells were digested and lysed by RIPA buffer supplemented with sodium orthovanadate, phenylmethylsulfonyl fluoride (PMSF), protease and phosphatase inhibitors (Thermo Scientific). Protein concentration was determined by a BCA assay (Pierce). All lysates were separated by SDS‐PAGE, and then, the separated proteins were transferred to nitrocellulose membrane (Thermo Scientific). The membranes were blocked with 5% bovine serum albumin in PBS with 0.1% Tween 20 at 37°C for 2 h. The transferred membrane was blotted with the following primary antibodies: WT1 (1:5000, ab89901; Abcam); E‐cadherin (1:2000, ab40772; Abcam); HSP90 (1:2000, ab13492; Abcam); USP5 (1:2000, ab155993; Abcam); Flag (1:1000, ab125243; Abcam); HA (1:1000, AE008; Abclone Biotechnology); β‐actin (1:5000, ab8227, Abcam). All secondary antibodies are conjugated with horseradish peroxidase (HRP). Signals were detected by chemiluminescence reagents (Thermo Scientific).