## 2 Results
### E3 ligase APC/C\({}^{\text{Cdh1}}\) Destabilizes FAH Protein  
To analyze the factors regulating the FAH protein levels, we screened a panel of E3 ligases available in our lab that might be involved in the reduction of FAH protein levels by Western blot analysis. Flag-TRCP1, Flag-TRCP2, HA-Cbl/Flag-Grb2 and Flag-Cdh1 were co-transfected with a constant amount of Myc-FAH in HEK293 cells. Among several E3 ligases, Flag-Cdh1 was associated with a significant reduction in FAH protein level (Figure 3A). Next, we transfected Flag-Cdh1 in a dose-dependent manner along with a constant amount of Myc-FAH construct in HEK293 cells. The dose-dependent increase in Cdh1 expression proportionally decreased FAH protein level in a dose-dependent manner, indicating that Cdh1 destabilizes FAH protein (Figure 3B). Similarly, at endogenous levels, we observed that a gradual increase in Cdh1 expression caused a proportional decrease in endogenous FAH expression in HHSteCs (Figure 3C). Next, we wished to analyze the effect of overexpression of Cdh1 on FAH protein ubiquitination. To this end, we co-transfected HEK293 cells with Myc-FAH, HA-ubiquitin and Flag-Cdh1 and performed immunoprecipitation analysis with anti-Myc antibody followed by immunoblotting with anti-HAantibody. The overexpression of Cdh1 led to increased high molecular weight smear of polyubiquitin chain conjugated with FAH protein (Figure 3D, lane 4) when compared with the absence of Cdh1 on FAH polyubiquitin (Figure 3D, lane 3). Thus, our results signify that APC/C\({}^{\text{Cdh1}}\) is an E3 ligase responsible for the destabilization and protein degradation of FAH protein. Figure 2: FAH undergoes proteolysis through 26S proteasomal pathway. **(A)** Exogenous Myc-FAH expression upon dose-dependent treatment of MG132 was determined in HEK293 cells through Western blot analysis with suitable antibodies. The Myc-FAH band intensity was estimated by ImageJ software and normalized with Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as control for graphical representation. Data were presented as the mean and standard deviation of three independent experiments. One-way ANOVA followed by [PERSON]’s post hoc test was used and \(p\)-values are indicated. **(B)** HEK293 cells were treated with dimethylsulfoxide as control or MG132 (7.5\(\upmu\)M) to check the exogenous Myc-FAH expression through Western blot analysis. The Myc-FAH band intensity was estimated by ImageJ software and normalized with GAPDH for graphical representation. Two-way ANOVA followed by [PERSON]’s post hoc test was used and \(p\)-values are indicated. **(C)** Myc-FAH was transfected individually or with HA-ubiquitin into HEK293 cells for immunoprecipitation with Myc antibody followed by immunoblotting with HA or Myc antibody. Reciprocal immunoprecipitation was performed with HA antibody, followed by immunoblotting with HA or Myc antibody. **(D)** Myc-FAH was transfected with HA-ubiquitin and cells were treated with MG132 in HEK293 cells to evaluate the effects on the ubiquitination and proteolysis of Myc-FAH. Cell lysates were immunoprecipitated with Myc antibody followed by immunoblotting with HA or Myc antibody. **(E)** Tandem Ubiquitin Binding Entities (TUBEs) assay for the ubiquitination of FAH proteins were conducted in Human hepatic stellate cells (HHSteCs) in the presence or absence of IL-1B keeping only agarose beads as control. Cell lysates were immunoprecipitated with TUBE antibody followed by immunoblotting with specific anti-FAH antibody. Lane1 is Input, lane 2 is control only agarose beads, lane 3 (without IL-1\(\beta\)) IP with Tubes and immunoblot with anti-FAH antibody and lane 4 (with IL-1\(\beta\)) IP with Tubes and immunoblot with anti-FAH antibody. Figure 3: E3 ligase APC/C\({}^{\text{Cdh1}}\) destabilizes FAH protein. (**A**) Screening E3 ligases for FAH. Several ligases were transfected transiently along with Myc-FAH in HEK293 cells and analyzed through Western blotting.