Mitophagy activity of <i>ISG15</i>^+/+ and <i>ISG15</i>^-/- BMDM.

<p><b>(A)</b><i>ISG15</i>^+/+ or <i>ISG15</i>^-/-BMDMs treated or not with IFN (500 units/ml, 16 hours) were infected (1 PFU/cell) with VACV at the times indicated. Cellular lysates were analyzed by 12 or 7.5% SDS-PAGE followed by transfer to nitrocellulose membranes. The expression of ATG3, ATG5, ATG7, LC3B and β-actin (protein loading control) was detected by western blotting using specific antibodies. Molecular weights are indicated. <b>(B)</b> Altered levels of mitochondrial dynamism in ISG15^-/- BMDM. <i>ISG15</i>^<i>+/+</i> or <i>ISG15</i>^<i>-/-</i> BMDMs treated or not with IFN (500 units/ml, 16 hours) were infected (1 PFU/cell) with VACV at the times indicated. Cellular lysates were analyzed by 12 or 7,5% SDS-PAGE, transferred to nitrocellulose membranes and the expression of OPA1, SDHA and tubulin (protein loading control) were detected by Western blot using specific antibodies. MWs are indicated. <b>(C)</b> Subcellular COX4 and p62 localization. Uninfected <i>ISG15</i>^+/+ or <i>ISG15</i>^-/- BMDMs treated or not with IFN (500 units/ml, 16 hours) were fixed and stained using a specific COX4 and p62 antibodies post-infection. 4 ',6-diamino-2-fenilindol (DAPI) was used to stain DNA (blue). Cells were visualized by confocal immunofluorescence microscopy. The images show representative fields (×73 magnification). <b>(D)</b> Validation of mitochondrial protein in cytoplasmic or mitochondrial protein extracts from BMDM from IFN-treated or not <i>ISG15</i>^+/+ or <i>ISG15</i>^-/- mice (20 μg). Proteins were fractionated by 12% SDS-PAGE, transferred to nitrocellulose membranes, and incubated with anti-TOMM20, anti-COX4, anti-Parkin or anti-VDAC proteins. Total protein loaded into the gel is visualized after Ponceau staining. The asterisks are signal specific for the protein molecular weight marker used. Molecular weights are indicated.