Fig 1: TRIM21 induces SUCLG2 degradation through the K63‐linkage ubiquitin lysosomal pathway. A) BEAS‐2B, A549, HCC827, and H1299 cells were treated with 20 µg mL−1 CHX and collected at the indicated time. The degradation rate of SUCLG2 was tested by western blotting (left‐hand panels). Relative SUCLG2 expression was analyzed using ImageJ (right‐hand panel). Data represent the average of three independent experiments (mean ± SD). ***p < 0.001. B) CHX (20 µg mL−1) was added to A549 cells for 24 h, and CQ (20 µm) or MG132 (20 µm) was added at the same time. The expression of SUCLG2 was detected using western blotting. C) pcDNA3.1‐His‐SUCLG2 was overexpressed in A549 cells treated with MG132 (20 µm) or CQ (20 µm) for 24 h. Co‐IP and western blotting were used to detect the ubiquitination level of SUCLG2. D) The indicated plasmids were transfected into A549 cells treated with CQ (20 µm) for 24 h. Co‐IP was used to detect the K63‐linkage ubiquitination level of SUCLG2. E) The E3 ligase for SUCLG2 identified by mass spectrometry is shown. F) The interaction between SUCLG2 and TRIM21 was detected using co‐IP in A549 cells transfected with pcDNA3.1‐His‐SUCLG2 and pCMV‐HA‐TRIM21. G) A549 cells were transfected with pCMV‐HA‐TRIM21, and western blotting was used to detect the expression of the indicated proteins. H) A549 cells transfected with or without pCMV‐HA‐TRIM21 were treated with CHX (20 µg mL−1) for the indicated times. The protein stability of SUCLG2 was detected by western blotting. I) The protein levels of SUCLG2 and TRIM21 were detected by western blotting in LUAD cell lines and human bronchial epithelial cell line BEAS‐2B. J) The protein levels of SUCLG2, TRIM21, and SIRT5 in 8 LUAD tissues were detected by western blotting (upper panels). T: tumor tissue. Protein correlation analysis was performed using ImageJ software and Prism 8 (bottom panels). K) The indicated plasmids were transfected into A549 cells. Co‐IP and western blotting were used to detect the K63 ubiquitination level of SUCLG2. L) The ubiquitination sites of SUCLG2 predicted by PhosphoSitePlus. M) A549 cells were transfected with the indicated plasmids, and the ubiquitination of SUCLG2 was detected by co‐IP and western blotting. N) A549 cells were transfected with the indicated plasmids, then treated with CHX (20 µg mL−1) for 0, 3, 6, 9, 12, and 24 h. The degradation rate of SUCLG2 was detected by western blotting.