Fig. S2.
Analysis of 5-HT2B/ERG-Fab complex formation using aSEC and a pull-down assay. (A) aSEC UV absorption traces of 5 µg of 5-HT2B/ERG (black), 5 µg Fab (blue), or 5 µg 5-HT2B/ERG incubated with 5 µg Fab for 1 h on ice (red). Upon binding to 5-HT2B/ERG, no more free Fab is detected. Complex formation is confirmed according to a nearly doubled peak volume of the receptor species and a shift to shorter retention time indicating an increase in molecular weight (Inset). (B) SDS/PAGE analysis of pull-down experiments using 1 µg of 5-HT2B/ERG, 1 µg of His-tagged Fab, or 1 µg of 5-HT2B/ERG mixed with His-tagged Fab. All samples were incubated on ice for 1 h, bound to TALON resin, and eluted using 20 mM Hepes pH 7.4, 500 mM NaCl, 0.05/0.01% DDM/CHS. Loaded material (Load), flow through (FT), and eluate (E) were analyzed using SDS/PAGE. Analysis confirms 5-HT2B/ERG-Fab complex formation as His-tagged Fab can capture 5-HT2B/ERG which otherwise does not bind the resin.