Anti usp7

A549 and H1299 cells were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.2% Triton X-100 for 15 min, blocked with 5% bovine serum albumin, and then incubated with anti-WDR79 (Bethyl Laboratories, Inc., Montgomery, TX, USA), anti-p53, anti-MDM2 (Santa Cruz) or anti-USP7 (Bethyl Laboratories, Inc.) antibodies at 4 °C overnight, followed by a DyLight 594-conjugated or DyLight 488-conjugated secondary antibody (ImmunoReagents, Inc., Raleigh, NC, USA). Cells were stained with 2-(4-Amidinophenyl)-6-indolecarbamidinedihydrochloride (DAPI) (Beyotime Biotechnology, Haimen, China) for 10 min, and the images were acquired with a confocal-microscope.

DUB plasmids and HA-ubiquitin were purchased from Addgene (Cambridge, MA, USA). USP7^WT (USP7 wild-type) and USP7^C223S (USP7 catalytic mutant) were cloned into a pFLAG-CMV-4 vector. GFP-tagged USP7 constructs (WT or mutants) were kindly provided by Prof. Jing Liu (Central South University, Changsha, China). Additionally, pcDNA3-Myc-ICN1 and pcDNA3-FLAG-ICN1 were obtained from Prof. Hudan Liu (Wuhan University, Wuhan, China).
The following antibodies were used in this study: anti-cleaved NOTCH1 (Val1744) and anti-β-Actin (CST, Danves, MA, USA); anti-USP7 (Bethyl Laboratories, Montgomery, TX, USA); anti-NOTCH1 and anti-GFP (Santa Cruz, Dallas, TX, USA); anti-HA and anti-Myc epitope tag (MBL, Nagoya, Japan); anti-FLAG (M2) (Sigma-Aldrich, Louis, MO, USA); and anti-HRP-conjugated secondary antibody (Millipore, Bedford, MA, USA). All antibodies were diluted according to the manufacturer’s recommendations.
The USP7 inhibitor P22077 [1-(5-((2,4-difluorophenyl)thio)-4-nitrothiophen-2-yl) ethanone] was obtained from EMD Millipore (EMD Millipore, Billerica, MA, USA). MG132 and cycloheximide (CHX) were purchased from Sigma-Aldrich.

Sigma Chemical Co. provided honokiol, cycloheximide and MG132 (St. Louis, MO, USA), and R&D system supplied recombinant human recombinant TRAIL and z-VAD-fmk (Minneapolis, MN, USA). Enzo Life Sciences provided lactacystin (Ann Arbor, MI, USA). The primary antibodies were as follows: Cell Signaling Technology supplied anti-PARP, anti-cleaved caspase-3, anti-Bcl-xL, anti-DR5, anti-CHOP, and anti-UCHL1 (Beverly, MA, USA). Sigma Chemical Co. supplied anti-actin (St. Louis, MO, USA). Enzo Life Sciences provided anti-pro-caspase-3 and anti-c-FLIP (San Diego, CA, USA). BD Biosciences provided anti-Bim and anti-XIAP (San Jose, CA, USA). Abcam supplied anti-DR4 (Cambridge, MA, USA). R&D system supplied anti-survivin (Minneapolis, MN, USA). Santa Cruz Biotechnology provided anti-Mcl-1, anti-Bcl-2, anti-cIAP2, anti-ATF4, anti-Ub, anti-Cbl, anti-Itch, anti-USP14, anti-USP33, anti-OTUB1, anti-TRABID, and anti-STAMBPL1 (St. Louis, MO, USA). Bethyl Laboratories Inc provided anti-USP7 and anti-USP8 (Montgomery, TX, USA). Novus Biologicals supplied anti-USP53 (Centennial, CO, USA). Abnova provided anti-USP9X (Taipei City, Taiwan). The siRNAs were as follows: GFP (control) siRNA (Bioneer, Daejeon, Korea), DR5 siRNA (Invitrogen, Carlsbad, CA, USA), and STAMBPL1 siRNA (Santa Cruz Biotechnology, St. Louis, MO, USA). STAMBPL1 plasmid was a gift from Dr. H.C. Kang (Ajou University, Suwon, Korea).

The following antibodies were used: anti-PAR, mouse monoclonal, Trevigen, 4335-MC-100; anti-PAR (10H), mouse monoclonal, Enzo Life Sciences, ALX-804-220; anti-ubiquitin, mouse monoclonal, Santa Cruz, sc-8017; anti-His tag, mouse monoclonal, Abcam, ab18184; anti-His tag, rabbit monoclonal, Cell signaling, 12698; anti-TOP1, mouse monoclonal, BD Biosciences, 556597; anti-dsDNA, mouse monoclonal, Abcam, ab27156; anti-FLAG, mouse monoclonal, Sigma Aldrich, F1804; anti-FLAG, rabbit polyclonal, Sigma Aldrich, F7425; anti-TDP1, rabbit polyclonal, Bethyl Laboratories, A301-618A; anti-PARP1 (F2), mouse monoclonal, Santa Cruz, sc-8007; anti-PARG, rabbit monoclonal, Cell Signaling, 66564; anti-γH2AX, rabbit polyclonal, Cell Signaling, 66564; anti-BrdU, mouse monoclonal, Abcam, ab8152; anti-USP7, rabbit polyclonal, Bethyl Lab, A300-033A; Rabbit TrueBlot: anti-rabbit IgG HRP, Rockland, 18-8816-31; Mouse TrueBlot ULTRA: anti-mouse Ig HRP, Rockland, 18-8817-31; anti-Rabbit IgG, Alexa Fluor 568, Thermo Fisher, A-11011; anti-Mouse IgG, Alexa Fluor 488, Thermo Fisher, A-28175.