Anti ub 10201 2 ap

The following antibodies were used: anti-GAPDH (5174, Cell Signaling Technology), anti-SLC7A11 (26864-1-AP, Proteintech), anti-p53 (10442-1-AP, Proteintech), anti-GPX4 (A11243, ABclonal Technology), anti-ACSL4 (A6826, ABclonal Technology), anti-FTL (ab69090, Abcam), anti-NRF2 (M200-3, Medical Biological Laboratories), anti-ATF3 (P18847, Cusabio), anti-ATF4 (P18848, Cusabio), anti-HA (M180, Medical Biological Laboratories), anti-DDDDK (M185, Medical Biological Laboratories), anti-ub (10201-2-AP, Proteintech), anti-RND2 (GXT56070, GeneTex), anti-RND3 (GTX81316, GeneTex) and anti-Mouse IgG (A7028, Beyotime Biotechnology). Several anti-RND1 antibodies in the market has been tried which showed the poor sensitivity, and they were prone to non-specific reactions with other RND members, such as RND2 and RND3. We selected the latter part of the RND1 sequence (CSLSKRLLHLPSRSE) to predict the antigen peptide and synthesized an anti-RND1 antibody by Genscript (Nanjing, China). The specificity of RND1 antibody was verified (Additional file 1: Fig. S1B). DMSO was purchased from Servicebio (WD2650, Servicebio). The apoptosis inhibitor Z-VAD-FMK (S7023), the ferroptosis inhibitor Ferrostatin-1 (S7243), the necrosis inhibitor Necrosulfonamide (S8251) and the ferroptosis inducer Erastin (S7242) were purchased from Selleck (USA).

Huh7, HepG2, and HEK293T cells were cultured in Dulbecco’s modified Eagle medium (Bio-Channel, China) supplemented with 10% fetal bovine serum (Bio-Channel, China) and penicillin-streptomycin. Flag-tagged RIG-I, MDA5, MAVS, TBK1, IRF3, HA-tagged WT-Ub, K48-Ub, and K63-Ub plasmids were kept in our laboratory. HA-tagged USP15 were created by cloning the CDS (coding sequence) region of USP15 into the pcDNA3.1-HA vector. Plasmids, miRNA mimics, and inhibitors were transfected into cells using M5 Hiper Lip.2000 (Mei5bio) according to the manufacturer’s instructions. All the antibodies used in this research were listed as follows: anti-Flag (M20008) and anti-HA (M20003) antibodies were purchased from Abmart; anti-RIG-I (20566–1-AP), anti-USP15 (14354–1-AP), anti-Ub (10201–2-AP), and anti-GAPDH (60004–1-Ig) were purchased from Proteintech; anti-K48-Ub (8081), anti-K63-Ub (5621), and anti-Phospho-IRF3 (37829) were obtained from Cell Signaling Technology.