1. CHGB preferentially located to the outside of the vesicles and mapping its membrane interacting fragments.
CHGB preferentially located to the outside of the vesicles and mapping its membrane interacting fragments. (A) Schematic diagram of the Ca2+-release assay. 1.0 μM Indo-1 (black star) in the outside would fluoresce stronger when Ca2+ ions (red dots) are released from vesicles and bound to Indo-1 (green stars). Excitation: 340 nm. Emission: 410 nm. (B) Positive control for the Ca2+-release assay. Type 1 IP3R (a calcium release channel) protein was purified from rat cerebellum and reconstituted into egg PC vesicles with 1.0 mM CaCl2. The vesicles were changed into a Ca2+-free buffer before being used. The calcium release was induced by adding 1.0 μM IP3. β-OG was used to release all calcium at the end, and the data were normalized against the maximum signal. The experiments were repeated more than 4 times with similar results. (C). A diagram showing the direct measurement of chloride flux using an Ag/AgCl electrode. (D) Ca2+-loaded vesicles treated with trypsin while the fluorescence of Indo-1 was monitored as in (B). The experiments were performed at pH 7.4 (black) and 5.5 (grey). (E) Coomassie-blue–stained SDS–PAGE for the gradient fractions after the vesicle floatation experiments of different CHGB fragments as GST fusion proteins. (F) Schematic depiction of the light-scattering–based flux assay.
Gaya P Yadav et al. LSA 2018;1:e
© 2018 Yadav et al.