Fig. 5: 1D movement protects MDA5 from abnormal activation. | Cell Research

Fig. 5: 1D movement protects MDA5 from abnormal activation.

From: ATP-dependent one-dimensional movement maintains immune homeostasis by suppressing spontaneous MDA5 filament assembly

Fig. 5

a The frequency of MAVS-CARD recruitment by 100 nM MDA5, MDA5(R337G) and MDA5(M854K) in the absence or presence of 100 nM LGP2 (mean ± SD; n = number of dsRNA molecules). b Representative kymographs showing the recruitments of AF647-MAVS-CARD (100 nM) by Cy3-MDA5, Cy3-MDA5(R337G) and Cy3-MDA5(M854K) (100 nM) in the absence of LGP2. Cy3-MDA5 protein is shown in green and AF647-MAVS-CARD is shown in red. c Representative kymographs showing the recruitments of AF647-MAVS-CARD (100 nM) by Cy3-MDA5, Cy3-MDA5(R337G) and Cy3-MDA5(M854K) (100 nM) in the presence of LGP2 (100 nM). Cy3-MDA5 protein is shown in green and AF647-MAVS-CARD is shown in red. d Column plots of relative IFNβ mRNA levels showing the MDA5 signaling activity under various conditions. 293∆RIG-I with MDA5(R337G) and LGP2 co-overexpression (MDA5(R337G)+LGP2), 293∆RIG-I with MDA5(M854K) and LGP2 co-overexpression (MDA5(M854K)+LGP2) and 293∆RIG-I with MDA5 and LGP2 co-overexpression (MDA5+LGP2) were stimulated with dsRNA substrates of different lengths. e Relative IFNβ mRNA levels in relation to dsRNA length. f Column plots of relative Ifnβ, Ifit1 and Cxcl10 mRNA levels showing the MDA5 signaling activity in MEFs under various conditions. g Immunoblotting showing the phosphorylation of TBK1 and IRF3 in MEFs cells. All single-molecule studies were performed at least two separate times. All cell-based assays were repeated independently at three times with similar results. Error bars represent SEM between measurements, centered on the mean of a single experiment.

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