Fig 1: Continued FIGURE 5MuV-induced expression of type 1 interferons (IFN). (A) IFN expression in SC. SC were isolated from three-week-old WT, Axl -/-, Mer -/- and Axl -/- Mer -/- mice, and were infected with 1.0 MOI of MuV for the specified durations (h). Relative mRNA levels of Ifna (left panel) and Ifnb (right panel) were determined by real-time qRT-PCR. (B) IFN expression in LC. WT, Axl -/-, Mer -/- and Axl -/- Mer -/- LC were infected with MuV for the indicated durations. Relative mRNA levels of Ifna and Ifnb were determined. (C) IFN secretion. SC and LC were infected with 1.0 MOI of MuV for 48 h. The secretion of IFNA and IFNB in culture media were determined using ELISA. (D) IFN expression in vivo. MuV (1 × 107 PFU) in 10 µl of PBS was injected into each testis of 8-week-old WT and Axl -/- Mer -/- mice (n = 3 mice each group). Total RNA was extracted from the testis. Relative RNA levels of Ifna (left panel) and Ifnb (right panel) were determined by real-time qRT-PCR. Data are shown as the mean ± SEM of three experiments. * p < 0.05, ** p < 0.01.
Fig 2: Role of IFN signaling in MuV replication. (A) MuV-NP RNA levels. WT and Axl -/- Mer -/- SC, and LC were infected with 1.0 MOI of MuV in the absence (-) and presence (+) of 2 µg/ml neutralizing antibodies against type 1 IFN receptor (anti-IFNAR). MuV-NP RNA levels were determined using real-time qRT-PCR at 48 h after MuV infection. (B) MuV-NP protein levels. SC (left panels) and LC (right panels) were treated as described in (A). MuV-NP protein levels were determined using Western blot. (C) MuV loads in culture media. SC and LC were treated as described in (A). MuV loads in culture media were measured by TCID50 assay. (D) MuV replication in type 1 IFN receptor knockout (Ifnar1 -/-) cells. SC and LC were isolated from three-week-old Ifnar1 -/- and WT mice. Cells were infected with 1.0 MOI of MuV. MuV-NP RNA (left panel) and protein (right panels) levels were determined at 48 h post infection. (E) Effect of LDC1267, an inhibitor of AXL and MER, on MuV replication. WT and Ifnar1 -/- SC and LC were infected with 1.0 MOI of MuV in the presence of LDC1267. The cells were infected with MuV in the absence of LDC1267 served as the controls (Ctrl). MuV-NP RNA (left panels) and protein (right panels) levels were determined using real-time qRT-PCR and Western blot at 48 h post infection. (F) MuV loads. SC and LC were treated as described in (E). MuV loads in culture media were determined using TCID50 assay. Images of Western blot represent at least three independent experiments. Data are presented as the mean ± SEM of three experiments. * p < 0.05, ** p < 0.01.
Fig 3: Role of AXL and MER in MuV replication. (A) Expression of AXL and MER receptors. SC and LC were isolated from 3-week-old wide-type (WT), Axl knockout (Axl -/-), Mer knockout (Mer -/-), and Axl/Mer double knockout (Axl -/- Mer -/-) mice. The expression of AXL and MER was examined by Western blot. (B) MuV binding. SC and LC with the indicated genotypes were incubated with 100 MOI of MuV for 1 h on ice. MuV-NP RNA (upper panels) and protein (lower panels) levels were determined by real-time qRT-PCR and Western blot, respectively. (C) MuV internalization. SC and LC were incubated with 100 MOI of MuV for 1 h at 37°C. MuV-NP RNA (upper panels) and protein (lower panels) levels were determined. (D) MuV replication. SC and LC were infected with 1.0 MOI of MuV. MuV-NP RNA (upper panels) and protein (lower panels) levels were determined at 48 h after infection. (E) Intracellular MuV. WT and Axl -/- Mer -/- SC (left panels) and LC (right panels) were infected with MuV as described in (D). Intracellular MuV-NP (red) was localized by IF staining at 48 h. Cellular plasma and nuclei were visualized using IF for a-tubulin (green) and DAPI (blue) counterstaining, respectively. (F) MuV loads in culture medium. SC and LC were infected with MuV as described in (D). Virus loads in culture media were measured by TCID50 assay at 48 h after infection. (G) MuV replication in vivo, MuV (1 × 107 PFU) in 10 µl of PBS was injected into each testis of 8-week-old WT and Axl -/- Mer -/- mice (n = 3 mice each group). Total RNA was extracted from the testis and relative RNA level of MuV-NP was determined by real-time qRT-PCR. Images represent at least three independent experiments, scale bars, 50 µm. Data are presented as the mean ± SEM of three experiments. ns, not significant; * p < 0.05, ** p < 0.01.
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