## 2 Results
### FAH undergoes Proteolysis through the 26S Proteosomal Pathway  
To investigate whether FAH is regulated by the UPS degradative pathway, we estimated the FAH protein level in the presence or absence of the proteasomal inhibitor MG132. To this end, Myc-FAH was transfected in HEK293 cells and treated with increasing concentrations of proteasomal inhibitor MG132 for 8 h. We observed that MG132, which blocks 26S proteasome activity, increased FAH protein levels by accumulating proteins in a dose-dependent manner in the cells (Figure 2A). Next we wished to check whether the accumulation of FAH protein occurs due to post-translational modifications or transcriptional regulation. To this end, we performed quantitative real-time RT-PCR (qRT-PCR) for the Myc-FAH transfected samples treated with MG132 in a dose-dependent manner. The results showed that there were no significant changes in the mRNA level of FAH upon MG132 treatment, signifying that the accumulation of FAH protein is mainly due to the blockage of 26S proteasomalomal degradation system by MG132 (Supplementary Figure S3). To further validate proteasomalomal regulation of FAH, we performed translation shut off experiment in the presence or absence of MG132 at varied time points. Briefly, HEK293 cells were transfected with constant amount of Myc-FAH and treated with constant amount of either MG132 or DMSO as controls (7.5 uM). These cells were harvested at different time points and subjected to Western blot analysis. Our results indicate a significant increase in the stabilization of FAH protein in MG132 treated cells as compared with the DMSO treated cells (Figure 2B). The increase in FAH protein due to treatment with proteasome inhibitor MG132 led us to speculate that the UPS might be involved in regulating FAH protein turnover. To investigate whether FAH protein undergoes ubiquitination, we co-transfected Myc-tagged-FAH and HA-tagged-ubiquitin in HEK293 cells and performed an immunoprecipitation assay using an anti-Myc antibody followed by immunoblotting with an anti-HA antibody. Our results showed a characteristic high molecular weight smear of polyubiquitin molecules conjugated to FAH protein in the cells co-transfected with the FAH and ubiquitin construct, which indicated that FAH protein undergoes polyubiquitin (Figure 2C, lane 2).  
We next examined the effect of MG132 on FAH ubiquitination. The treatment with MG132 of the co-transfected cell cultures for 8 h resulted in the increased accumulation of polyubiquitinated FAH protein (Figure 2D, lane 4) as compared to the untreated samples (Figure 2D, lane 3). Furthermore, to validate whether FAH is undergoing endogenous ubiquitination, we performed TUBEs assay that has high affinity probe for ubiquitated proteins [22]. This was carried out in HHSteCs in the presence or absence of Interleukin-1 beta (IL-1\(\beta\)), keeping only agarose beads as control. Upon stimulation of IL-1\(\beta\), FAH protein undergoes rapid ubiquitination (Figure 2E, lane 4) when compared to non-stimulated cells (Figure 2E, lane 3). Thus, our results suggest that FAH protein interacts with ubiquitin molecules and undergoes polyubiquitin through UPS.