Figure 7. Functional Validation of USP13 within the ERAD pathway.
(A, B) HCIPs with Pfam domains associated with the ubiquitin-proteasome pathway found in association with Dubs (A). The number of HCIPs with the indicated Pfam domains found associated with particular Dubs is shown by the rainbow scale. The number of Dubs with HCIPs containing the indicated Pfam domains (orange bars) as well as the number of HCIPs containing the indicated domains (blue bars) are shown in (B).
(C) TSCs corresponding to VCP/p97 identified within immune-complexes of indicated Dubs.
(D) Schematic representation of USP13 and select HCIPs known to associate with VCP and their annotated Pfam domains.
(E) TCRαGFP cellular fluorescence levels after knockdown of indicated genes. Two siRNA oligos were used against each gene. Fluorescence levels are relative to control siRNA transfection (siCK). For western blots confirming knockdown see Figure S14
(F) Histograms of TCRαGFP fluorescence after knockdown of indicated genes.
(G) Cell viability, as measured by cellular ATP levels, was measured 72 hours after siRNA transfection and 48 hours after addition of 0.3μg/mL tunicamycin to the growth media. All values are relative to the amount viability measured after control siRNA transfection (siCK), Error bars represent the SEM of triplicate measurements. *p-value<0.05, **p-value<0.01 as determined by Student’s t-test.