Pr 619

Cells were lysed with RIPA lysis buffer containing 3 deubiquitylase inhibitors — 1,10-phenanthroline (P0221, Tokyo Chemistry Industry), N-ethylmaleimide (E0136, Tokyo Chemistry Industry), and PR-619 (HY-13814, MedChemExpress) — for the immunoprecipitation experiments and the analysis of ubiquitylated proteins by anti-multiubiquitin antibody. One to two milligrams of whole-cell lysates were used for immunoprecipitation and coimmunoprecipitation of AhR using the anti-AhR antibody. Briefly, the anti-AhR antibody in (NH₄)₂SO₄ was coupled to the pre-equilibrated Protein G Dynabeads in NaH₂PO₄ at 37°C overnight, following the manufacturer’s instructions. The AhR-coupled beads were then added to each sample and incubated on an orbital shaker at 0.5 g overnight at 4°C. After washing 3 times for 5 minutes each with the assay buffer, the beads were eluted with electrophoresis sample buffer for SDS-PAGE, followed by Western blot analysis.

Vx3 protein was obtained and covalently linked to α-Al₂O₃ nanoparticles to generate α-Al₂O₃-Vx3 nanoparticles according to our previous reports (12 (link), 13 (link)). 4T1/WT cells and 4T1/EPB cells were treated with 200 nM bortezomib (Millennium Pharmaceuticals, USA) and 20 mM NH₄Cl (Sigma, USA) for 9 h. The cells were collected and lysed in RIPA lysis buffer (Millipore, USA) containing protease inhibitors (MedChemExpress, USA), phosphatase inhibitors (MedChemExpress, USA), and PR-619 (MedChemExpress, USA). The cell lysate was reacted with α-Al₂O₃-Vx3 nanoparticles under stirring for 12 h at 4°C to generate α-Al₂O₃-UPs nanovaccine (named UP-nanovaccine) according to our previous study (13 (link), 20 (link)). The precipitates (UP-nanovaccine) were collected by centrifugation (12,000 g, 30 min, 4°C), and the supernatant was collected as unbound lysate, followed by the detection of ubiquitin protein levels in the three samples using Western blotting. The covalently linked product UP-nanovaccine was collected by centrifugation, and the number of UPs enriched by α-Al₂O₃-Vx3 was evaluated by collecting and calculating the difference between the number of UPs in the supernatant before and after the reaction by BCA Protein Assay Kit (Beyotime Biotechnology, China) according to the protocol of the manufacturer.

PR-619 was obtained from MedChemExpress (Junction, NJ, USA) and cisplatin from Merck Millipore (Billerica, MA, USA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Merck Millipore. For Western blot analysis, antibodies against cleaved caspase-3 (#9661), cleaved caspase-8 (#9496), B cell lymphoma (Bcl)-2 (#15071), phospho-Bcl-2 (#2824), phospho-p53 (#9284), caspase-4 (#4450), phospho-Janus kinase (JNK) (#9255), and c-Myc (#18683) were procured from Cell Signaling Technology (Danvers, MA, USA). The antibodies against β-actin (#109) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, #100118) were supplied by GeneTex (Irvine, CA, USA); those against α-tubulin (sc-5286), P21 (sc-6264), P27 (sc-1641), and JNK (sc-571) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). For IHC, the USP14 antibody (#MA5-32821) was purchased from Invitrogen, the USP21 antibody (#17856-1-AP) was purchased from Proteintech (Chicago, IL, USA), and the c-Myc (#ab32072) antibody was purchased from Abcam (Cambridge, UK).