Fig. 8: Gp78ΔG2BRc shows reduced ubiquitination compared to Gp78 and a further reduction in presence of VHH1_2G2 and SBP-His6-G2BR^AUP1; CUE-G2BR^AUP1 reduces Gp78 activity.

A Cartoon representing the region of Gp78ΔG2BRc (aa309-573) appended to GST for purification (top). SEC profiles show a mixture of a 2-fold molar excess of Ube2G2 over GST-Gp78ΔG2BRc (red) along with unbound Ube2G2 (green). The corresponding fractions of the mixture are shown below in an SDS PAGE gel. B Anti-ubiquitin immunoblot of ubiquitination assays performed with GST-Gp78, and GST- Gp78ΔG2BRc in the presence and absence of a 3-fold molar excess of VHH1_2G2, or unrelated VHH_SMT3 over Ube2G2 for the indicated time points. C Anti-ubiquitin immunoblot of ubiquitination assays performed similar to (B) using isolated G2BR constructs appended to SBP-His6 tags. D Anti-ubiquitin immunoblot of ubiquitination assays with GST-Gp78 in the presence and absence of a 3-fold molar excess of CUE-G2BR^AUP1, SBP-His6-G2BR^{AUP1 or Gp78} over Ube2G2 at indicated time points. E SEC profile of the ubiquitination assay sample at the 30-minute time point with a 3-fold molar excess of CUE-G2BR^AUP1 (red) indicated by an arrow in D. Profiles of CUE-G2BR^AUP1 (black) and Ube2G2 (green) are also shown. SDS PAGE analysis of the ubiquitination assay SEC fraction (red) is displayed to the right, with the major eluted species indicated at the bottom.