Sc 166882

Conventional protocols were used for IB with the details available elsewhere. The following primary antibodies were used for IB: anti-HERPUD1 (1:1000, ab150424, Abcam, Cambridge, UK), anti-GCLC (1:1000, ab190685, Abcam), anti-CBS (1:1000, ab140600, Abcam), anti-CTH (1:1000, ab189916, Abcam), anti-SHMT2 (1:1000, #33443, Cell Signaling Technology (CST), Boston, MA, USA), anti-GSS (1:1000, #ab124811, Abcam) or GSS (H-7) (1:500, sc-166882, Santa Cruz, Heidelberg, Germany), anti-MDM2 (1:1000, ab259265, Abcam), anti-SYVN1 (1:1000, #14773, CST), anti-GAPDH (1:1000, #5174, CST). The membranes were incubated with anti-rabbit IgG, HRP-linked antibody (1:2000, #7071, CST) or anti-mouse IgG, HRP-linked antibody (1:2000, #7076, CST) at room temperature for one hour, and visualized using Clarity Western ECL substrate. The relative expression levels of proteins were determined by densitometry using ImageJ software and were normalized to housekeeping protein.

Cell lysates were incubated with the corresponding antibodies overnight at 4 °C, followed by incubation with protein A/G beads. Subsequently, the beads were collected after washing with 1 × PBS and double-distilled water, and an appropriate amount of 1 × loading buffer was added for IB. The antibodies used for co-IP were anti-GSS (1:200, #ab124811, Abcam) or GSS (H-7) (1:50, sc-166882, Santa Cruz), anti-MDM2 (1:200, ab259265, Abcam), anti-SYVN1 (1:200, #14773, CST) and anti-ubiquitin (1:200, #3936, CST).

Total proteins were isolated from frozen kidney tissue or cultured cells using RIPA lysis buffer (R0010, Solarbio, Beijing, China) containing protease and phosphatase inhibitor cocktail (04693116001, Roche). Protein samples were separated in a 10% SDS page gel and transferred onto the PVDF membrane (R1SB07718, Millipore). After the blocking using 5% skimmed milk for 1 h at room temperature and three times washing with 1×PBST, the membrane was incubated with primary antibodies at 4°C overnight and then with the corresponding secondary antibodies with a dilution of 1:4000 for 1 h at room temperature. Proteins were visualized by enhanced chemiluminescence (ECL, 32209, Thermo Fisher Scientific). Primary antibodies against β-actin (4970S, Cell Signaling, 1:1000 dilution), GPX4 (ab125066, Abcam, 1:1000 dilution), GSS (SC-166882, Santa Cruz, 1:500 dilution), GSR (SC-133245, Santa Cruz, 1:500 dilution), JAK2 (3230S, Cell Signaling, 1:1000 dilution), phospho-JAK2 (3771S, Cell Signaling, 1:500 dilution), STAT3 (9139S, Cell Signaling, 1:1000 dilution), phospho-STAT3 (9145S, Cell Signaling, 1:1000 dilution). Secondary antibodies against anti-mouse HRP-linked antibody (7076S, Cell Signaling, 1:5000 dilution), anti-rabbit HRP-linked antibody (7074S, Cell Signaling, 1:5000 dilution).