Mg132

For mechanistic analysis, we used the NF-κB pathway inhibitor BAY 11-7082 (s2913; Selleck Chemicals, Shanghai, China) at a final concentration of 5 μM for 6 h. Additionally, we used the 26S proteasome inhibitor MG132 (s1748; Beyotime Biotechnology) at a final concentration of 10 μM for 5 h.

For proteasome inhibition assays, cells were transfected with the indicated plasmids (Figures 4 and 5B). At 48 h after transfection, the 26S proteasome inhibitor MG132 (s1748; Beyotime Biotechnology) was added at a final concentration of 10 μM for 5 h, after which samples were collected. Cells were lysed in lysis buffer, and cell debris was pelleted by centrifugation at 12,000 rpm for 10 min at 4°C. Supernatants were collected for IP. IP was carried out using whole cell lysates (approximately 200 μg protein) with 4–10 μg antibody and 20 μL protein A/G agarose (P2012; Beyotime Biosciences). The cell lysates were precleared with 20 μL agarose A/G beads by rocking for 1 h at 4°C. Beads were removed, and appropriate antibodies were added. Samples were then incubated with 20 μL agarose A/G beads by rocking for 4–6 h at 4°C, and lysates were then added, followed by incubation with rocking overnight at 4°C. The immune complexes were collected by centrifugation, followed by washing in cell lysis buffer before analysis. The levels of IκB-α ubiquitylation were evaluated by immunoprecipitation of IκB-α using anti-IκB-α antibodies followed by anti-HA immunoblotting.

For co-immunoprecipitation, 293T cells transfected with Flag‐FBXW7 and HA-DLL1 recombinant plasmids were homogenized in NP-40 Lysis Buffer (P0013F; Beyotime, Shanghai, China) containing MG-132 (S1748; Beyotime, China) and deubiquitinase inhibitor with anti‐Flag (F7425, 1:1,500; Sigma, USA) or anti‐HA (51064‐2‐AP, 1:1,000; Proteintech, USA) antibody at 4°C overnight, followed by coupling with A/G agarose beads for 4 h. Afterward, the immunoprecipitates were subjected to the detection of western blot again.

Three dyes, ThT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and DAPI, and two antibodies, the mouse anti-PrP monoclonal antibody 3F4^{41 (link),48 (link)} and a 10-nm gold-labeled anti-mouse antibody^{7 ,9 (link),57 (link)}, were purchased from Sigma-Aldrich (St. Louis, MO). Sarkosyl and PK were obtained from Amresco (Solon, OH). Alexa 546-conjugated fluorescent secondary antibody^{7 ,9 (link),57 (link)}, the proteasome inhibitor MG132, and the ROS probe DCFH-DA (2′,7′-dichlorofluorescein diacetate) were purchased from Beyotime (Nantong, China), respectively. Ni-Sepharose was obtained from GE Company (Pittsburgh, PA). All other chemicals used in this study were of analytical grade and were produced in China.

IS (purity > 99%) was dissolved by dimethyl sulfoxide (DMSO). The DMSO concentration in all drug-treated cells was less than 0.1 %. MTT, 5-FU and Insulin were purchased from Sigma (St Louis, MO). MG-132 was purchased from Beyotime (Haimen, China). CHX was purchased from Biovision (San Francisco, USA). All antibodies were purchased from Cell Signaling Technology (Danvers, MA).

Nutlin-3a and Erlotinib were purchased from Selleck chemicals (Houston, TX, USA). MG132 was from Beyotime Biotechnology (Shanghai, China). Chloroquine was from Abcam (Cambridge, UK), and all other chemicals were of analytical grade and purchased from Sigma Chemical (Burlington, USA). For in vitro and in vivo studies, Nutlin-3a was reconstituted in dimethyl sulfoxide (DMSO) and 2% DMSO/1% hydroxyethyl cellulose/0.2% Tween-80, respectively.