Fig 2: Protein–protein association between TRIM21 and endoglin. (A–E) Co-immunoprecipitation of TRIM21 and endoglin. A,B. HUVEC monolayers were lysed and total cell lysates (TCL) were subjected to SDS-PAGE under reducing (for TRIM21 detection) or nonreducing (for endoglin detection) conditions, followed by Western blot (WB) analysis using antibodies to endoglin, TRIM21 or β-actin (A). HUVECs lysates were subjected to immunoprecipitation (IP) with anti-TRIM21 or negative control antibodies, followed by WB analysis with anti-endoglin (B). C,D. CHO-K1 cells were transiently transfected with pDisplay–HA–Mock (Ø), pDisplay–HA–EngFL (E) or pcDNA3.1–HA–hTRIM21 (T) expression vectors, as indicated. Total cell lysates (TCL) were subjected to SDS-PAGE under nonreducing conditions and WB analysis using specific antibodies to endoglin, TRIM21, and β-actin (C). Cell lysates were subjected to immunoprecipitation (IP) with anti-TRIM21 or anti-endoglin antibodies, followed by SDS-PAGE under reducing (upper panel) or nonreducing (lower panel) conditions and WB analysis with anti-TRIM21 or anti-endoglin antibodies. Negative controls of appropriate IgG were included (D). E. CHO-K1 cells were transiently transfected with pcDNA3.1–HA–hTRIM21 and pDisplay–HA–Mock (Ø), pDisplay–HA–EngFL (FL; full-length), pDisplay–HA–EngEC (EC; cytoplasmic-less) or pDisplay–HA–EngTMEC (TMEC; cytoplasmic-less) expression vectors, as indicated. Cell lysates were subjected to immunoprecipitation with anti-TRIM21, followed by SDS-PAGE under reducing conditions and WB analysis with anti-endoglin antibodies, as indicated. The asterisk indicates the presence of a nonspecific band. Mr, molecular reference; Eng, endoglin; TRIM, TRIM21. (F) Protein–protein interactions between TRIM21 and endoglin using Bio-layer interferometry (BLItz). The Ni–NTA biosensors tips were loaded with 5.4 µM recombinant human TRIM21/6xHis at the N-terminus (R052), and protein binding was measured against 0.1% BSA in PBS (negative control) or 4.1 µM soluble endoglin (sEng). Kinetic sensorgrams were obtained using a single channel ForteBioBLItzTM instrument.

Fig 3: A working model of GAC acetylation in NSCLC. A working model depicting the molecular mechanism of HDAC4 mediated GAC deacetylation and TRIM21 mediated GAC ubiquitination to regulation tumorigenesis in NSCLC.

Fig 4: Trim21 ubiquitinates FIP200 for degradation. (A,B) Co-IP assay demonstrated interaction between endogenous FIP200 and Trim21. Anti-FIP200 Ab (A) or anti-Trim21 Ab (B) was used for immunoprecipitation. The mutual pulldown was detected by immunoblot. (C) Ubiquitination assay indicated Trim21 overexpression increased FIP200 ubiquitination level. HeLa cells were transfected with His-Ub and indicated plasmids. (D) Effect of the K1133R mutation on Trim21-induced FIP200 degradation was determined by immunoblot. HeLa cells were transfected with indicated plasmids. Here, a 3-fold amount of flag-FIP200 plasmid was transfected compared to the amount of K1133R mutant. Flag-FIP200 and K1133R mutant were quantified and normalized to GAPDH. (E) Ubiquitination assay showed that K1133R mutation reduced FIP200 Trim21-mediated ubiquitination. HeLa cells were transfected with His-Ub and indicated plasmids.

Fig 5: NLRP3 is required for inflammasome activation downstream of TRIM21 A–CPrimed HMDM were treated with MCC950 (1 μM) for 30 min before stimulation overnight with AdV (50,000 pp/cell) and h9C12 (20 μg/ml) or IVIg (20 mg/ml) or 10 μM Nigericin. IL‐1β (A) or TNF (B) or LDH release (C) was measured in cell supernatants. Data show average ± s.d. of two independent donors and are representative of three independent experiments.DWT, NLRP3, ASC or caspase‐1‐deficient THP‐1s were stimulated as in (A) and IL‐1β in the supernatant measured by ELISA. Data are representative of two independent experiments.EHMDM were primed with 10 μg/ml pI:C for 2 h, treated with 100 μM VX‐765 for 30 min before stimulation for 16 h as in (A). IL‐1β or LDH release was measured in cell supernatants. Data show average ± s.d. of two independent donors.FPrimed HMDM were treated with KCl as indicated for 1 h, before being stimulated for a further 6 h as in (A). IL‐1β and TNF were measured in cell supernatants by ELISA. Data are representative of three independent donors.GPrimed HMDM were stimulated with AdV and 20 μg/ml h9C12 or 10 μM Nigericin for 3 h and then immunostained for ASC or intracellular antibody. Co‐localisation was assessed by confocal microscopy. Data are representative of two independent donors.