Journal: ACS Omega
Article Title: Covalent Rpn13-Binding Inhibitors for the Treatment of Ovarian Cancer
Figure Lengend Snippet: RA183 binding to RPN13 is stronger than RA190. (A) Chemical structures of RA9, b-AP15, RA190, RA183, and RA183B, in which a biotin moiety (blue) is linked to RA183, and likewise for RA190B. (B) Purified 19S RP (200 ng) was labeled with RA183, RA190, RA183B, or RA190B (20 μM) alone or in the presence of competitor RA183 and RA190 (100 or 200 μM) for 1 h at 37 °C. After labeling, equal aliquots were boiled in sodium dodecyl sulfate (SDS) sample buffer, separated by SDS-polyacrylamide gel electrophoresis (PAGE), transferred to a poly(vinylidene difluoride) (PVDF) membrane, probed with streptavidin peroxidase, and developed with chemiluminescence for the recognition of biotinylated proteins. (C, D) Cell lines from cancers of different tissue origins were lysed in Mammalian Protein Extraction Reagent (M-PER). In addition, Escherichia coli transduced with a plasmid expressing 6His and T7 tag-labeled RPN13 or irrelevant antigen and induced with isopropyl β- d -1-thiogalactopyranoside (0.1 mM, 2 h) were lysed in B-PER. Cell lysates (40 μg of protein) were treated with 5 μM RA183B (L) or not (C) for 45 min at 4 °C and subjected to SDS-PAGE, transferred to PVDF membrane, and probed with horseradish peroxidase (HRP)–streptavidin. The asterisk corresponds to ∼42 kDa RPN13 band.
Article Snippet: Antibodies for Western Blot analysis were obtained by the following commercial sources: anti-p21(WAF1), Bax (Cell Signaling Technology, Danvers, MA), antiubiquitin (SC8017), anti-IkB-α (C-15), anti-ATF-4 (SC-200) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-PARP (BD Pharmingen, San Diego, California), (Stressgen Corp., Victoria, BC, Canada), antitubulin (Sigma, St. Louis, MO), anti-actin, anti-ADRM1 (anti-RPN13) (Sigma-Aldrich), antiubiquitin, Lys48 (05-1307) (Millipore), streptavidin Dynabeads (Invitrogen), HRP–streptavidin (Invitrogen), peroxidase-linked antimouse immunoglobulin G (IgG), and antirabbit IgG (GE Healthcare U.K. Ltd., U.K.) and utilized at the concentration recommended by the manufacturer.
Techniques: Binding Assay, Purification, Labeling, Polyacrylamide Gel Electrophoresis, Protein Extraction, Transduction, Plasmid Preparation, Expressing, SDS Page