Fig. 3
RidL binds Vps29 but not Vps29L152E in vitro. a For size-exclusion chromatography (SEC), RidL (132 kDa) was mixed in a molar ratio 1:1.5 with Vps29 (21 kDa) or Vps29L152E (21 kDa). SDS-PAGE and Coomassie Brilliant Blue staining of (A) input (ratio 1:1.5), (B) SEC elution fractions 9–13 ml (elution volume RidL or complex), and (C) SEC elution fractions 15.5–17.5 ml (elution volume Vps29 or Vps29L152E) were analyzed. SEC elution profiles are shown, analyzed fractions are labeled and elution peak elution volumes are indicated. The peak shift for the RidL–Vps29 complex is 0.2 ml relative to RidL alone. b For surface plasmon resonance (SPR) measurements, biotinylated RidL was used as ligand and Vps29 as analyte. Concentration series: red, 0 nM; brown, 50 nM; green, 150 nM; blue, 450 nM; cyan, 1.35 μM. c Response units (RU) obtained at binding equilibrium of each Vps29 concentration shown in b are plotted against concentration (nM). An equilibrium constant KD of 400 nM was calculated from the fit as indicated in the Materials and Methods section
