Fig. 3: HERC2 regulates protein levels of the USP20-ULK1 axis.

A Schematic representation of the USP20-ULK1 axis. ULK1 protein levels are regulated post-translationally by ubiquitylation-dependent protein degradation (left panel). The deubiquitylating enzyme USP20 interacts with ULK1, catalyzing its deubiquitylation and thereby enhancing ULK1 stability (right panel). HEK293T cells (B) or U2OS (C) were transfected with either a negative control siRNA (NC) or a siRNA targeting HERC2. Subsequently, the expression levels of the indicated proteins were assessed by immunoblot analysis. The intensity bands corresponding to USP20 and ULK1 were quantified and subsequently normalized using clathrin heavy chain (CHC) protein levels as a loading control. The results are presented as fold changes relative to the control condition. Plots display the mean ± standard error of the mean (SEM). Representative results are shown for experiments that were independently repeated at least three times, and individual data points for each independent experimental repetition are represented as individual dots on the graphs. Significance was determined using unpaired Student’s t-test. Significance levels: *p < 0.05; **p < 0.01. D Control cells and HERC2 knocked-down cells were fractionated into cytosol (cyto.) and membrane (mem.) using 0.015% digitonin. Immunoblotting of α-TUBULIN (cytosolic control) shows the successful separation of the cytosol and membrane. Data are representative of two independent experiments.