FIGURE 2.

Bag6 associates with the ER membrane via binding the UbxD8-gp78 complex. A, purification of Bag6, Trc35, or Ubl4A was performed using HEK293 cells expressing the indicated FLAG-tagged proteins. As a negative control, cells transfected with a control empty vector were used. The purified complexes were analyzed by SDS-PAGE and Coomassie Blue staining. B, immunoblotting confirms the interaction of Bag6 with UbxD8. Cells transfected with the indicated plasmids were lysed, and proteins immunoprecipitated with FLAG beads were analyzed by immunoblotting. LE, long exposure; S.E., short exposure. C, as in B, except that cells expressing FLAG-tagged UbxD8 were used. D, interaction of the endogenous Bag6 complex with UbxD8. Whole cell extracts were subject to immunoprecipitation by the indicated antibodies. The asterisk indicates IgG. E and F, UbxD8-gp78 complex is required for membrane association of Bag6. E, CLM extracts were treated with protein A beads containing either control IgG or the indicated antibodies. After depletion, proteolipisomes were re-formed and used in binding experiments with purified Bag6. Proteins bound to the membrane pellet fractions were analyzed by immunoblotting. Where indicated, a buffer control was included to assess the levels of background binding to residual Bio-Beads present in the samples. The numbers indicate band intensity. LE, long exposure. F, graph shows the quantification of the experiment in E. Error bar indicates the mean of the two independent experiments.