Trim21

Proteins from LV tissues and cells were extracted in RIPA buffer and quantified with a BCA protein assay kit. Then, the extracts were separated with SDS-PAGE and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). Next, the membrane was blocked with 5% nonfat milk containing 0.1% PBST for 1 hour at room temperature and subsequently incubated with primary antibodies overnight at 4°C. After washing with PBST and incubating with the appropriate secondary antibody, the bands were visualized using ECL Western Blotting Detection Reagent (Tanon, Shanghai, China). Primary antibodies used in the study are listed as follows: TRIM21 (Abcam), F4/80 (Abcam), Bcl-2 (CST), Bax (CST), γ-H2AX (Abcam), iNOS (CST), and Arg1 (Proteintech Group, Chicago, IL, USA) phosphatidylinositol 3 kinase (PI3K, CST), p-PI3K (CST), protein kinase B (AKT, CST), p-Akt (CST), β-actin (CST), Vinculin (Santa Cruz) and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH, CST).

The specific reagents included the primary antibodies against AKT, POU2F1, and TRIM21 (Abcam, London, UK); against PI3K^p100α, γ-H₂AX, 53BP1 and p-AKT^Ser473 (Cell Signaling Technology, Danvers, MA, USA); against α-tubulin, BCL-2 and BAX (Proteintech, Chicago, USA); horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Beyotime, Shanghai, China). GSK2795039, 3-MA and DADS (oil, ≥98%, and 1.008 g/mL) were purchased from Sigma, (Saint Louis, Missouri, USA), and the DADS was fully dissolved in Tween 80 and diluted at 1:100 in physiological saline and stored in a -20°C freezer. Additionally, 740Y-P, LY49002, MG132, AG activator 1 and cycloheximide as well as AV-153, an antimutagenic molecule were obtained from Selleckchem, Houston, USA and MedChenExpress, Monmouth Junctio, USA, respectively.