Fig. 5

TRIM25 binding to the RNA augments TRIM25 ubiquitination. a Western blot analysis of HeLa cells transfected with EGFP-TRIM25 or EGFP-TRIM25ΔRBD. Lane 1 shows mock transfected control. Lanes 2 and 3 represent results of transfection with increasing amounts of plasmid coding for EGFP-TRIM25 (250 and 500 ng). Lanes 4 and 5 represent results of transfection with increasing amounts of plasmid coding for EGFP-TRIM25ΔRBD (1000 and 2000 ng). b Quantification of western blot results shown in (a). The values show the ratios between the intensity of modified versus main bands. The results are derived from three independent biological replicates and the mean and standard deviations are shown. c Western blot analysis of TRIM25 KO HeLa cells transfected with plasmids coding for T7-TRIM25ΔRING (250 ng) or T7-TRIM25ΔRING_3′-UTR (500 ng) as well as increasing amounts of plasmids coding for EGFP-TRIM25 or EGFP-TRIM25ΔRBD. Lane 1 shows T7-TRIM25ΔRING control. Lanes 2 and 3 represent results of transfections with T7-TRIM25ΔRING and increasing amounts of EGFP-TRIM25 (250 and 500 ng). Lanes 4 and 5 show results of transfection with T7-TRIM25ΔRING and increasing amounts of EGFP-TRIM25ΔRBD (1000 and 2000 ng). Lane 6 shows T7-TRIM25ΔRING_3′-UTR control. Lanes 7 and 8 represent results of transfections with T7-TRIM25ΔRING_3′-UTR and increasing amounts of EGFP-TRIM25 (250 and 500 ng). Lanes 9 and 10 show results of transfection with T7-TRIM25ΔRING_3′-UTR and increasing amounts of EGFP-TRIM25ΔRBD (1000 and 2000 ng). d Quantification of western blot results shown in (c). The values show the ratios between the intensity of modified versus main bands. The results are derived from three independent biological replicates and the mean and standard deviations are shown