Dominant-negative ERAD components inhibit HShh-C degradation. (A) Cells stably expressing the HShh-HA precursor were transfected with a catalytically inactive Myc-tagged Hrd1 (Hrd1-C291A) or with an empty vector. The fate of HShh-HA was followed after addition of cycloheximide (CHX), by SDS-PAGE and immunoblotting for HA; immunoblotting for p97 served as a loading control. All samples were also analyzed by SDS-PAGE followed by immunoblotting for Myc (Hrd1-C291A) and for glyceraldehyde 3-phosphate dehydrogenase (GAPDH; loading control). The right graph shows the quantification of the HShh-C in the experiment. n = 4 time points. (B) As in A, but with transfection of either wild-type p97 (p97-WT), a catalytically inactive p97 mutant (p97-QQ), or with an empty vector. The HA blot was stripped and reprobed with anti-GAPDH antibodies (loading control). Endogenous and overexpressed p97 were detected on the same gel by immunoblotting with anti-p97 antibodies. n = 3 time points. (C) HShh-HA was transiently expressed in 293T cells together with dominant-negative Ubc6e (Ubc6e-C91S), control vector, or UbxD8-GFP. Greater than 90% of the cells showed a strong GFP signal by live-cell fluorescence microscopy (not depicted). Protein synthesis was inhibited with cycloheximide, and the fate of HShh-HA was followed by SDS-PAGE and immunoblotting with HA antibodies. Ponceau S staining of the blot is shown to demonstrate the loading of equal amounts of protein. Molecular masses are given in kilodaltons.