Fig 1: MAGI1 suppresses IFN signaling in ECs. (A) HUVECs were transfected with either siCont or siMAGI1. After 48 h of transfection, total RNA was extracted then qRT-PCR was performed. Relative changes in Magi1, Mx1, Ifnb1, Ifna1, IfngI, Stat1, Stat5a, and Stat5b expression were calculated using the comparative Ct (2−ΔΔCt) method. Ct values of each target gene were normalized to that of the Gapdh (26). Each group passed the Shapiro-Wilk normality test, then an unpaired student t-test was performed using the Prism software (GraphPad Software). The graph shows mean ± SD (n = 11). **p < 0.01, *p < 0.05. (B) HUVECs were transfected with either siCont or siMAGI1. After 48 h of transfection, total RNA was extracted then qRT-PCR was performed. Relative changes in Oas2 mRNA expression were calculated using the comparative Ct (2−ΔΔCt) method. Ct values of Oas2 were normalized to that of the Gapdh (26). Each group passed the Shapiro-Wilk normality test, then an unpaired student t-test was performed using the Prism software (GraphPad Software). The graph shows mean ± SD (n = 3). **p < 0.01. (C) HUVECs were transfected with either siCont or siMAGI1. After 48 h of transfection, cell lysates were analyzed by immunoblotting with each specific antibody as indicated. The graphs represent densitometry data from 3 independent gels, one of which is shown in the left panel. Each group passed the Shapiro-Wilk normality test, then an unpaired student t-test was performed using the Prism software (GraphPad Software). The graph shows mean ± SD, n = 3, **P < 0.01, and * P < 0.05. Uncropped figures were provided in the supplements. (D) HUVECs were transfected with either siCont or siMAGI1. After 48 h of transfection, conditioned medium was collected and levels of IFNβ were measured. Each group passed the Shapiro-Wilk normality test, then an unpaired student t-test was performed using the Prism software (GraphPad Software). The graph shows mean ± SD, n = 4–6, *P < 0.05. (E,F) HUVECs were transfected with either the pCMV-MAGI1 or the pCMV-2B backbone construct (control) (20). After 24 h of transfection, total RNA was extracted then qRT-PCR was performed. Relative changes in Magi1 (E) and Ifnb1 (F) mRNA expression were calculated using the comparative Ct (2−ΔΔCt) method. Ct values of each target gene were normalized to that of the gapdh (26). At least, one of the groups did not pass the Shapiro-Wilk normality test, we performed an unpaired t-test with Welch's correction using the Prism software (GraphPad Software). The graph shows mean ± SD, n = 5, **P < 0.01. (G) HUVECs were transfected with either the pCMV-MAGI1 or the pCMV-2B backbone construct (control) (20). After 24 h of transfection, the cells were treated with IFN (200 U/mL) for 6 h, and immunoblotting was performed with each specific antibodies as indicated. The graph (lower) represents densitometry data from 4 independent gels, one of which is shown in the top panel. Each group passed the Shapiro-Wilk normality test, then one-way ANOVA followed by Turkey's multiple comparisons test was performed using the Prism software (GraphPad Software). The graph shows mean ± SD (n = 4). *p < 0.05.
Supplier Page from Abcam for Anti-STAT1 antibody