## 2 Results
### APC/CC\({}^{\text{Cdh1}}\) Regulates FAH Protein Half-Life  
We wished to check the half-life of FAH protein and the effect of the overexpression of Cdh1 on FAH protein half-life. To analyze FAH protein turnover in the presence and absence of Cdh1, we performed cycloheximide assay in HEK293 cells transfected with Flag-Cdh1 or sgRNA-1 targeting Cdh1 along with ectopically expressed Myc-FAH. Our results showed that the half-life of FAH is about 6 h (Figure 6B). Interestingly, the knockout of Cdh1 led to the extension of FAH protein half-life when compared with samples transfected with Myc-FAH alone (Figure 6C). In contrast, Myc-FAH protein half-life was significantly reduced in the presence of Cdh1 when compared with samples transfected with the original Cdh1.  
Figure 4: APC/CC\({}^{\text{Cdh1}}\) is a negative regulator of FAH protein stability. (**A**) Schematic of the RNA-guided engineered nuclease targeting the human Cdh1 gene with designed sgRNA1 and sgRNA2 that target sequences in exon 10. Yellow boxes represent exons. Red box indicates the positions of sgRNAs targeting the top strand. Target sequences are represented in black and PAM sequences are represented in red. (**B**) T7E1 assays were performed in HEK293 cells to determine the cleavage efficiency of sgRNA-1 and sgRNA-2 by transfecting Cas9, sgRNA-1 and sgRNA-2. The samples were loaded in 2% agarose gel. The two bands in the sgRNA-1 and sgRNA-2 results indicate the cleaved size of DNA and are indicated by arrows along with the indel percentages measured by band intensity using ImageJ software. Untransfected HEK293 cells were used as a control. The marker is shown for size reference. (**C**) HEK293 cells were transiently transfected with sgRNA-1 and sgRNA-2 targeting Cdh1 along with ectopically expressed Myc-FAH to check exogenous protein levels. (**D**) HHSteCs were transfected with sgRNA-1 and sgRNA-2 targeting Cdh1 to check endogenous FAH protein levels using FAH specific antibody and endogenous Cdh1 was detected using anti-Cdh1 antibody. (**E**) Reconstitution effect of Cdh1 on exogenous FAH protein level in Cdh1-depleted HEK293 cells. (**F**) Reconstitution effect of Cdh1 on endogenous FAH protein level in Cdh1-depleted HHSteCs. The FAH band intensity for (**C**–**F**) was estimated by ImageJ software and normalized with GAPDH 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.  
with Myc-FAH alone (Figure 6D). Thus, the depletion of Cdh1 is responsible for the extension of FAH protein half-life.  
Figure 5: APC/C\({}^{\text{Cdh1}}\) co-localizes and interacts with FAH. (**A**) Co-localization of FAH and Cdh1 was performed in HHSteCs by immunostaining with specific antibodies. DAPI was used for nuclear staining (scale bar: 10 \(\upmu\)m) and the quantification for colocalization coefficients were done using Fiji/ImageJ software. To quantify the degree of co-localization in between the fluorochromes, the Pearson’s correlation coefficient (r) was used. The classical Pearson coefficient of the pixel-intensity correlation of colocalization obtained from the coloc2 analysis were mentioned. (**B**) Myc-tagged FAH and Flag-tagged Cdh1 were co-transfected into HEK293 cells. Samples were immunoprecipitated using anti-Myc and immunoblotted using anti-Flag antibodies. GAPDH was used as the loading control. (**C**) Myc-tagged FAH and Flag-tagged Cdh1 were co-transfected into HEK293 cells. Samples were immunoprecipitated using anti-Flag and immunoblotted using anti-Myc antibodies using anti-Myc antibodies. GAPDH was used as the loading control. (**D**) Endogenous binding between FAH and Cdh1 proteins was performed in HHSteCs. Cell lysates from HHSteCs were immunoprecipitated with specific FAH or Cdh1 antibodies and immunoblotted with FAH or Cdh1 antibodies.