Figure 5.

Ub binding induces ALIX-V dimerization in vitro. (A) Shows MST experiment monitoring the oligomerization of ALIX-V in solution. Fluorescently labelled untagged ALIX-V at a constant concentration with increasing concentrations of non-labelled ALIX-V were used and changes in thermophoresis were measured (top). Hill analysis resulted in concentration dependent oligomerization; (bottom) oligomerization in the presence of Ub. (B) Cross-linking assay of ALIX-V. In ALIX-V concentration of 7 μM (below the EC₅₀), oligomerization is not detected (first lane from left). Increasing Ub concentrations 1.5, 4.0, 7.5, 15.0, 40.0 and 75.0 μM promote the oligomerization of ALIX-V (lanes 2–7). Cross-linking assay with 75.0 μM of L8E, I44E and V70D triple Ub mutant is shown (lane 8). Control with maximal Ub concentration but without ALIX-V is shown (lane 9). Control without cross-linker is shown at the bottom gel. (C) Cross-linking assay of ALIX-V in the EC₅₀ concentration (27.6 μM), oligomerization of ALIX-V is shown in the presence and absence of 75.0 μM Ub.