Additional file 8 of Lethal (2) giant discs (Lgd)/CC2D1 is required for the full activity of the ESCRT machinery

Additional file 8: Figure S8. LGD1 binding does not affect the conformation of CHMP4B in solution. (A) Schematic representation of cysteine-based crosslinking assay of CHMP4B. (B) Representative result of the assay performed three times independently. The percentages are the average of all three experiments. Coomassie-Blue staining of non-reducing SDS-PAGE gel of CHMP4Bredox. In solution, the majority of CHMP4B exists in the faster migrating closed conformation (73,9%). Nevertheless, a significant fraction (26,1%) exists in the slower migrating open conformation (lane 1). As expected, the addition of the reducing DTT (10 mM) abolishes the fraction of CHMP4Bredox existing in the closed form (lane 2). The fraction that exists in the closed form can be fixed by addition of Cu2+Phenanthroline, which forms intramolecular disulfide bonds between the two existing cysteines in the N- and C-terminus (lane 3). In Lane 4, CHMP4Bredox was first incubated with DTT for 20 min. at 37 °C to remove the fraction existing in the closed form. Then, the sample was incubated at room temperature for 30 min. before Cu2+Phenanthroline was added to catch the re-emerging fraction of closed form. 29,9% of CHMP4B has again adopted the closed conformation, indicating that CHMP4B is in a dynamic equilibrium between the open and closed conformation. Lanes 5–7: The addition of LGD1 does not affect the conformation of CHMP4B, indicated by the fact that the distribution of closed and open CHMP4B molecules is comparable to corresponding samples without LGD1 (lanes 1, 3, and 4).