Trim25 antibody

MG132 (HY-13259), Z-VAD-FMK (HY-16658B), erastin (HY-15763), 3-MA (HY-19312), Fer-1 (HY-100579), necrostatin-1 (HY-15760), cycloheximide (HY-12320) and RSL3 (HY-100218A) were obtained from MedChemExpress. JNJ-64619178 (S8624) and GSK3326595 (S8664) were purchased from Selleck Chemicals. Mouse anti-PD-1 antibody (BE0146) and IgG control (BE0089) were purchased from Bio X Cell. Recombinant murine IFN-γ (P6137) was purchased from Beyotime. PRMT5 (D5P2T), actin (8H10D10), and symmetric di-Methyl arginine motif (13222) were purchased from Cell Signaling Technology (Massachusetts, USA). KEAP1 antibody (10 503–2-AP), NRF2 antibody (16 396–1-AP), HMOX1 antibody (10 701–1-AP), V5 antibody (14 440–1-AP), HA antibody (51 064–2-AP), TRIM25 antibody (12 573–1-AP), FLAG antibody (20 543–1-AP), SLC7A11 antibody (26 864–1-AP), and GPX4 antibody (67 763–1-Ig) were purchased from Proteintech.

Tissue sections were deparaffinized and endogenous peroxidase was blocked with 3% H₂O₂. Sections were incubated in 5% albumin bovine V (BSA, Solarbio) for 1 h. The cells were washed three times with PBS and fixed with 4% paraformaldehyde for 15 min. After three washes with PBS, the cells were permeated with 0.1% Triton X-100 for 15 min. After three washes with PBS, the cells were incubated with 5% bovine serum albumin for 30 min at room temperature and then incubated with p21 antibody (1:300, Abcam, Cambridge, MA, USA), p16 antibody (1:200, Abcam), SFTPC antibody (1:200, Abclonal, Wuhan, China), Keap1 antibody (1:300, Proteintech, Wuhan, China), Nrf2 antibody (1:300, Cell Signaling Technology, USA), PCNA antibody (1:200, Proteintech), Ki67 antibody (1:200, Abways, Shanghai, China), or Trim25 antibody (1:200, Proteintech) at 4 °C overnight. The next day, after washing with PBS, fluorescein-labeled secondary antibodies (1:300, Abcam) for immunofluorescence were incubated. Nuclei were counterstained with DAPI (Invitrogen, USA). The samples were washed three times with PBS and photographed with fluorescence microscopy (Nikon, Japan).