Fig. 6

TRIM21 interacts with ANXA2 and triggers ubiquitination. A: ANXA2 expression in HUVECs with or without FGF19 treatment following CHX treatment for the indicated times. B: ANXA2 expression in HUVECs with or without the addition of Mg132 following CHX treatment for the indicated times. C: Co-IP was performed to analyse the interaction between ANXA2 and TRIM21 in HUVECs. IgG was used as a negative control. D: Representative images of colocalization of ANXA2 and TRIM21 in HUVECs. Red: ANXA2; Green: TRIM21. Pearson's R value of scatter plot analysis was calculated using ImageJ. E: ANXA2 and TRIM21 expression in HUVECs after FGF19 treatment was detected by immunofluorescence staining. F: The interference efficiency of siTRIM21 was assessed by western blotting in HUVECs. G: qRT-PCR was used to detect mRNA level of TRIM21 and ANXA2 with the transfection of NC or siTRIM21. H: Western blot was used to detect protein level of TRIM21 and ANXA2 with the transfection of NC or siTRIM21. I: ANXA2 expression in HUVECs transfected with NC or siTRIM21 following CHX treatment for the indicated times. J: HUVECs transfected with NC or siTRIM21 were immunoprecipitated with ANXA2 antibody and analysed by immunoblotting with the anti-ubiquitin antibody to examine ANXA2 ubiquitination. Whole-cell lysates were used for western blotting with an anti-TRIM21 or anti-ANXA2 antibody. K: Tube formation assays (top) and Transwell migration assays (bottom) were performed to measure tube formation and migration of shANXA2-HUVECs transfected with NC or siTRIM21. L, M: The relative tube length and number of migrated HUVECs were quantified. Data represent the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001