Fig. 4

MDA5-myc reduces the expression levels of LINE-1 ORF1p and ORF2p. A Schematic representation of LINE-1 expression cassettes in L1–1FH and L1–2TAP, both of which were generated with pcDNA6/myc-His B as the backbone vector. L1–1FH expresses ORF1p with a Flag and HA tag at the C-terminus, and L1–2TAP expresses ORF2p with a TAP tag at the C-terminus. B Bar chart showing relative protein levels of Western blotting results that indicate MDA5-myc’s effect on the ORF1p expressed from L1–1FH. HEK293T cells seeded in a 24-well plate were co-transfected with 1 μg of L1–1FH and 225 ng of control vector VR1012 or 25, 75, or 225 ng of MDA5-myc-expressing vector, and subjected to western blotting at 48 h post-transfection. C Bar chart showing relative protein levels of Western blotting results that indicate MDA5-myc’s effect on endogenous ORF1p protein levels. HEK293T cells seeded in a 24-well plate were co-transfected with 1 μg of L1–1FH and 225 ng of control vector VR1012 or 25, 75, or 225 ng of MDA5-myc-expressing vector and subjected to western blotting at 48 h post-transfection. D and E Bar charts showing relative protein levels of Western blotting results that indicate the effects of MDA5-myc or its mutants on the expression levels of ORF1p and ORF2p-TAP expressed from L1–2TAP. HEK293T cells seeded in a 24-well plate were co-transfected with 1 μg of L1–2TAP and 225 ng of control vector VR1012 or 25, 75, or 225 ng of one of the vectors expressing wild type MDA5-myc or its mutants. Transfected cells were subjected to western blotting at 48 h post-transfection. All bar charts in this figure were generated based on relative levels of ORF1p and/or ORF2p of three independent experiments, and are shown as means ± SEM. Presentative western bloting results for each panel are shown in Fig. S3