Fig. 6: DRP1 ISGylation affects its function.
A Cytosolic and mitochondrial fractions obtained from semi-permeabilised cells, transfected with indicated constructs, were immunoblotted against phospho-DRP1 (S616) and total DRP1; VINCULIN and VDAC1 served as controls for cytosolic and mitochondrial fractions, respectively. Note lower levels of phospho-DRP1 (S616) and DRP1 in DRP1K532R mitochondrial fractions. B Graph represents data from 3 independent experiments. **p ≤ 0.01, ***p ≤ 0.001, using unpaired 2-tailed Student’s t-test. Error bars,±SEM. C Cells transfected with indicated mCherry-tagged constructs and treated with MitoTracker Green FM were imaged in the slice 3D-SIM live mode. White boxes show insets. White arrowheads mark the mCherry and mitochondria localisation. Scale bar, 10μm. D Graph represents number of DRP1 puncta / 2μm length of mitochondria from 3 independent experiments. ***p ≤ 0.001 using unpaired 2-tailed Student’s t-test. Error bars,±SEM. ~50 mitochondria were analysed. E Cells transfected with indicated mCherry-tagged constructs and treated with MitoTracker Green FM were imaged under live-cell conditions. Scale bar, 20μm. F Box plot showing quantification of mitochondrial length; ~100 cells from 3 independent experiments were analysed. The central line and the plus (+) symbol in each box show the median and mean value, respectively. ns, not significant (p = 0.6), *p ≤ 0.05, ***p ≤ 0.001 using unpaired 2-tailed Student’s t-test. G Cytosolic and mitochondrial fractions generated as in panel A were immunoblotted against phospho-DRP1 (S616) and total DRP1; VINCULIN and VDAC1 served as controls for cytosolic and mitochondrial fractions, respectively. H Graph represents data from 3 independent experiments. ns, not significant (p = 0.7), **p ≤ 0.01, ***p ≤ 0.001 using unpaired 2-tailed Student’s t-test. Error bars,±SEM.
