Stub1

The hemisected kidneys were fixed with 10% formalin and then embedded in paraffin wax. After cutting into 4 μm-thick sections, the slides were stained with hematoxylin and eosin (HE) and evaluated under a light microscope. For immunohistochemistry (IHC) staining, antigen retrieval was performed after deparaffinization. Subsequently, endogenous peroxidase activity was blocked by 3% H₂O₂ and then incubated with 5% BSA for 30 min at 37°C. The sections were incubated with primary antibodies against STUB1 (Proteintech), VHL (Proteintech), and SOCS1 (Proteintech) overnight. Then, after incubation with HRP-conjugated secondary antibody, the proteins were visualized using DAB (Beyotime).

MTT, 3-methyladenin (3-MA), pifithrin-α (PFT-α), RPMI-1640, and other chemicals were purchased from Sigma-Aldrich. Gapdh, NCOA4, Bcl-2, and AKT antibody were obtained from EnoGene (Nanjing, China); antibodies EGFR, stub-1, SOD, MDM2, Cyt-C, LC3, Bax, and p-AKT were purchased from Proteintech Group Inc. (Wuhan, China). Antibodies p53, p-p53, and ferritin were purchased from Cell Signaling Technology (Massachusetts, USA); siRNA for MDM2 (stB0001232) and stub1 (stB0001238) were obtained from RiboBio (Guangzhou, China).

HK-2 cells or animal kidney tissue lysates were prepared in RIPA buffer (Beyotime Biotechnology, Shanghai, China). The protein concentrations were detected by the BCA method (Beyotime). Briefly, 40 μg of protein was subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Then, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, United States). After blocking with 5% non-fatty milk for 1 h at room temperature, the membranes were incubated with primary antibodies of STUB1 (Proteintech, Wuhan, China), VHL (Proteintech), SOCS1 (Proteintech), tubulin (Proteintech), Bcl-2 (Cell Signaling, Beverly, MA, United States), Bax (Cell Signaling), cleaved caspase-3 (Cell Signaling), and caspase-3 (Cell Signaling) at 4°C overnight. Then, the membranes were incubated with HRP-conjugated anti-rabbit secondary antibody (Cell Signaling) at room temperature for 1 h. The proteins were then visualized by incubating with a Chemiluminescence (ECL) Western Blot Kit (Thermo Fisher Scientific).