Figure S2.

AP180_ANTH Does Not Bind VAMP2, VAMP3, or VAMP8, Related to Figure 2

(A) GST Pull downs using His₆MycAP180_ANTH and the GST-fusion proteins indicated. Top panel: Coomassie blue stained gel. Lower panel: western blot probed with anti-myc. The lane adjacent to the molecular weight markers (MWM) is loaded with His₆MycAP180_ANTH only. The ANTH domain of AP180 does not bind to VAMP2, 3, and 8.

(B) Isothermal titration calorimetry of the binding of CALM_ANTH and AP180_ANTH to Ins(1,4,5)P₃ gave comparable binding affinities (K_D24 ± 5μM and K_D23 ± 3μM respectively) confirming both proteins were folded and functionally active. Data for CALM is translated by −1.2 μcal/s for clarity.

(C) Surface Plasmon Resonance of CALM_ANTH and AP180_ANTH also showed AP180_ANTH did not bind to GST VAMP8. The SNARE was coupled directly to a CM5 chip and the binding of the ANTH domain proteins was assayed by flowing 200 nM analyte (CALM_ANTH or AP180_ANTH) across the chip for 60 s at a flow rate of 30μl/min in 20 mM HEPES (pH 7.4), 150 mM NaCl, 4 mM DTT. Data were recorded on a Biacore 3000 (GE Healthcare) at 20°C and the binding of analyte to a mock channel (GST) has been subtracted.