Figure 6.
Doxo treatment induces complex formation between BAG6 and ESCRT proteins. (A) Immunoblot analysis of cell lysates and BAG6 immunoprecipitations of HEK293 cells that were either non-treated or treated with 1µM doxorubicin for the indicated time probed with specific antibodies against BAG6, the ESCRT proteins HRS, TSG101 and ALIX and actin as a loading control. The blot represents one out of two independent experiments. (B) Analysis of the interaction of BAG6 and TSG101 using the Yeast two hybrid method. BAG6, BAG6 without TSG101 binding motif (BAG6del with deleted PSAP/PTAP motifs at amino acid positions 471-475 and 496-499, see Figure S6A) and TSG101 were subcloned in pGADT7 and pGBT9 expression vectors transformed into yeast lines Y2H Gold and Y187 or control vectors and mated. NKp30 and BAG6 were used as a positive control for interaction analysis and experiments were performed in two independent runs. doxo, doxorubicin; kDa, kilodalton; DDO, Minimal Media Double Dropouts; QDO, Minimal Media Quadruple Dropouts (QDO)