Fig. 6: AVAN binds to TRIM25 directly and enhances the TRIM25-mediated K63-linked ubiquitination of RIG-I. | Cell Death & Differentiation

Fig. 6: AVAN binds to TRIM25 directly and enhances the TRIM25-mediated K63-linked ubiquitination of RIG-I.

From: Long noncoding RNA AVAN promotes antiviral innate immunity by interacting with TRIM25 and enhancing the transcription of FOXO3a

Fig. 6

A RNA pull-down of AVAN-associated proteins using biotinylated AVAN or antisense probes. Isolated proteins were resolved by SDS-PAGE followed by silver staining. B The list of proteins identified by MS in AVAN sense and antisense group, and particularly TRIM25 was pulled down by AVAN in BJ501-infected A549 cells but not by the AVAN antisense. C Pull-down western blot showing that AVAN can bind directly to TRIM25. D ChIRP followed by western blot shows that AVAN can bind to TRIM25. Exogenous (E) and endogenous (F) RIP of TRIM25 in BJ501-infected cells using anti-TRIM25 or anti-IgG antibodies. The relative enrichment fold of AVAN was calculated by qRT-PCR (**p < 0.01). TRIM25 co-immunoprecipitation with proteins from lysates of BJ501-infected A549 cells transfected with AVAN (G) or AVAN-siRNAs (H), followed by immunoblotting. Anti-TRIM25 and anti-RIG-I antibodies were used for immunoprecipitated. Immunoblot analysis of endogenous RIG-I ubiquitylation in BJ501-infected A549 cells transfected with AVANs (I) or AVAN-siRNAs (J). Anti-RIG-I and anti-Ub antibody were used for immunoprecipitated. K Immunoblot analysis of proteins immunoprecipitated with anti-Flag from lysates of BJ501-infected A549 cells transfected with AVAN, HA-Ub, and Flag-tagged RIG-I. L IFN-α (left) and IFN-β (right) expression upon AVAN transfection in A549 cells that were infected by BJ501 or not (MOI = 1) at 24 h post infection, and then individually knockdown RIG-I or TRIM25, analyzed by qRT-PCR (n = 3; means ± SEM; *p < 0.05; **p < 0.01). M AVAN pull-down western blot with lysates of A549 cells transfected with Flag, Flag-TRIM25, Flag-SPRY, Flag-B Box/CCD, or Flag-Ring. N Truncated AVAN pull-down truncates (upper panel) were obtained via in vitro transcription and incubated with BJ501-infected A549 lysates for RNA pull-down. O IFN-α (left) and IFN-β (right) expression analyzed by qRT_PCR upon AVAN or AVAN truncates transfection in A549 cells that were infected by BJ501 or not (MOI = 1) at 24 h post infection (n = 3; means ± SEM; # p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001).

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