Fig. 7.

Parkin promotes degradation of monoubiquitinated CHIP. a IB analyses of Parkin and autophagy-related proteins in LPS-primed WT or FAM73b KO BMDMs. b Park2 mRNA was detected by RT-QPCR. c Confocal microscopy analyses of Parkin and mitochondria colocalization.d FAM73b KO BMDMs were pretreated with Mdivi-1 for 12 h. The Parkin level was detected by IB. e Cytokine induction in Parkin KO BMDMs was measured by RT-QPCR. f IB analysis of CHIP in the cytoplasm and IRF1 protein level and modification. g, h Monoubiquitination of CHIP in the cytoplasm (g) and cytokine induction (h) were performed in park2-silenced BMDMs. i Parkin–CHIP interaction assays in HEK293T cells co-transfected with plasmids encoding the indicated genes. After treatment with MG132, whole-cell lysates were subjected to IP using anti-HA, followed by IB analysis of associated FLAG-CHIP using anti-FLAG. j HEK293T cells were transfected with total ubiquitin plasmid along with the indicated expression plasmids. FLAG-tagged CHIP was isolated by IP, followed by detection of polyubiquitinated CHIP by IB. The data are representative of three independent experiments. Scale bar in c is 10 μm.Two-tailed unpaired t-tests were performed; *P < 0.05