Mitochondrial Ubiquitin Ligase MARCH5 Promotes TLR7 Signaling by Attenuating TANK Action
Figure 1
Identification of MARCH5 in TLR7 signaling pathway.
A, HEK293T cells were transfected with HA-MARCH5 and then treated with the nonspecific control (N.C.) or MARCH5 siRNA (left panel). Raw264.7 cells were transfected with the negative control (N.C.) or MARCH5 siRNA (right panel). Cell lysates were immunoblotted with the indicated antibodies. B, HEK293T cells were transfected with HA-hMARCH5 and then treated with the nonspecific control (N.C.) or hMARCH5 siRNA (left panel). HEK293T cells were transfected with the negative control (N.C.) or hMARCH5 siRNA (right panel). Cell lysates were immunoblotted with the indicated antibodies. C and D, the nonspecific control (N.C.) or MARCH5 siRNA were transfected into Raw264.7 cells with NF-κB (C) or PRDIII-I reporter plasmids (D), respectively. Forty-eight hours after transfection, cells were stimulated with poly(I:C) (2 µg/ml, transfected), poly(I:C) (50 µg/ml, added to the culture medium), R837 (10 µg/ml), Loxoribine (50 µM) or LPS (100 ng/ml) for 6 h before luciferase assays were performed. A pTK-Renilla reporter was used to normalize data. E and F, the nonspecific control (N.C.) or hMARCH5 siRNA were transfected into HEK293T cells together with NF-κB (E) or IFNβ reporter plasmids (F), respectively. Forty-eight hours after transfection, cells were transfected again with RIG-I, MAVS, TRIF, MyD88 or IRAK1 for 16 h before luciferase assays were performed. A pTK-Renilla reporter was used to normalize data. G, HA-MARCH5 was transfected into HEK293T cells (upper panel) or HeLa cells (lower panel), which were then stained with an anti-HA antibody and imaged by confocal microscopy. The mitochondria were stained with MitoTracker. H, HEK293T cells were fractionated as shown in diagram (top panel). The corresponding fractions were analyzed by immunoblotting with the indicated antibodies (bottom panel). I and J, Raw264.7 cells were stimulated with R837 (10 µg/ml) or LPS (100 ng/ml), respectively, for the indicated time periods. The mRNA level of MARCH5 was measured by quantitative PCR (I), and the protein level of MARCH5 was measured by immunoblotting with an anti-MARCH5 antibody (J). Data from C–F and I are presented as means ± S.D. from three independent experiments. *, P<0.05; **, P<0.01.