Ab155095

Antibodies and reagents were acquired from designated suppliers:
TRIM26 (ab188017, Abcam), TRIM26 (27013-1-AP, Proteintech), TRIM26 (sc-393832, Santa Cruz Biotechnology), GPX4 (ab125066, Abcam), GPX4 (sc-166570, Santa Cruz Biotechnology), PLK1 (ab189139, Abcam), p-PLK1 (ab155095, Abcam), β-actin (ab8226, Abcam), K48-ubiquitin (ab140601, Abcam), K63-ubiquitin (12930, Cell Signaling Technology), anti-Flag (66008, Proteintech), anti-Myc (16286, Proteintech), p-S/T (61 G, abmart), p-S/T (612549, BD Biosciences), Anti-His (66005, Proteintech), anti-Flag (14793, Cell Signaling Technology), anti-Myc (2276, Cell Signaling Technology), Anti-His (12689, Cell Signaling Technology), anti-HA (3724, Cell Signaling Technology),
MDA (ab27642, Abcam), 4-HNE (ab48506, Abcam), MG132 (S1748, Beyotime), cycloheximide (HY12320, MedChemExpress), Chloroquine (HY-17589A, MedChemExpress), Onvansertib (HY-15828, MedChemExpress), MLN0905 (HY-15155, MedChemExpress), Recombinant Human PLK1 (ab271716, Abcam).

Cells were lysed in the lysis buffer and the protein concentration was measured using the BCA protein assay kit (Thermo Fisher Scientific). The 10 μg proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto the polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were blocked with 5% skim milk for 2 h and incubated with rabbit anti-MYBL2 (1:1000, ab76009, Abcam, Cambridge, UK), cyclin A2 (1:10000, ab32386, Abcam, Cambridge, UK), cyclin B1 (1:20000, ab32053). Abcam, Cambridge, UK), GAPDH (1:10000, ab180630, Abcam, UK) and Plk1 (1:1000, ab155095, Abcam, Cambridge, UK) overnight at 4°C. Then the membranes were cultured with secondary antibody goat anti-rabbit IgG H&L (ab205718, Abcam, Cambridge, UK) for 1 h at room temperature. Finally, the electrochemiluminescence kit (ECL; Pierce Biotechnology) was used for protein development.

Protein was extracted from EndoC-βH1 cells in radioimmunoprecipitation assay buffer (Sigma-Aldrich) supplemented with halt protease and phosphatase inhibitor Cocktail (Thermo Fisher Scientific). Protein samples were loaded onto NuPAGE 4–12% Bis–Tris Protein Gels (Thermo Fisher Scientific), resolved by electrophoresis and transferred onto nitrocellulose membranes. Membranes were incubated with the following primary antibodies: mouse monoclonal anti-β-actin antibody (Invitrogen, MA1-140; 1:20,000), rabbit monoclonal anti-phospho CHEK2 T68 antibody (Cell Signaling, 2197S; 1:1,000), mouse monoclonal anti-CHEK2 antibody (Cell Signaling, 3440T; 1:1,000), rabbit anti-eIF2α antibody (Cell Signaling, 5324; 1:1,000), rabbit anti-phospho eIF2α Ser51 antibody (Cell Signaling, 3597; 1:1,000), rabbit anti-α-tubulin antibody (Cell Signaling, 2144; 1:1,000), rabbit anti-phospho PLK1 T210 (Abcam, ab155095; 1:200) and mouse anti-PLK1 (Abcam, ab17056; 1:1,000). Primary antibodies were detected by fluorophore-conjugated secondary goat anti-rabbit (LI-COR IRDye 800CW, 926-32213; 1:15,000) and donkey anti-mouse (LI-COR IRDye 680RD, 926-32210; 1:15,000) using LI-COR Odyssey Imagers. Western blot images were analyzed with Image Studio software 5.2.5.

To confirm the differences in the expression of the NRGs between ccRCC and normal tissues, we performed experimental validation using specimens obtained from 5 patients with ccRCC who underwent radical nephrectomy or partial nephrectomy from June 2021 to December 2022 at Wuhan University Renmin Hospital. Our study was approved by the internal review board of Renmin Hospital of Wuhan University.
The non-tumor tissues and tumor tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut in 4-μm-thick sections. The sections were incubated overnight at 4^°C with primary antibodies for MYCN (1:150, Abcam, Inc., ab227822), tumor necrosis factor receptor-associated factor 2 (TRAF2) (1:150, Abcam, Inc., ab167163), Bcl-2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) (1:100, Abcam, Inc., ab205606), and polo-like kinase 1 (PLK1) (1:500, Abcam, Inc., ab155095), and with secondary antibody for 30 min at 37^°C. The sections were incubated with 3,3′-Diaminobenzidine (DAB) chromogen for 8 min and followed by hematoxylin and bluing reagent counterstaining. Under a microscope, five different fields of view were randomly selected to assess staining intensity. Two pathologists independently assessed the slides of immunohistochemical staining.