Atpγs

A549 and HeLa cells stably expressing a
GCaMP6s-3xNLS-P2A-mBeRFP-3xNLS reporter transgene were seeded into the wells
of a black 96-well glass bottom plate (Cellvis; #P96–1.5H-N) at a
density of 2 × 10⁴ cells per well 24 hrs before imaging.
Before the experiment, cells were washed 2 times with L-15 medium and
equilibrated to 50 µL of L-15 medium for 30 min at RT. Images were
acquired every 15 sec from two different fields of view for a period of 22
min. During the experiment, 20 µM of ATP (Sigma; #A26209),
ATPγS (Tocris; #4080), mipATP, AMP-PNP (Sigma; #A2647) or vehicle was
added in 50 µL of L-15 medium to the well prior to the 2-min
timepoint. (final concentration of 10 µM nucleotide).
Images were captured at RT (~26°C) using NIS-Elements (Nikon)
on an Eclipse Ti microscope (Nikon) equipped with a 20× Plan
Apochromat NA 0.75 air objective lens, a Clara CCD camera (Andor), and a
motorized stage. GCaMP6s and mBeRFP fluorescence were excited with a LED
light source (Lumencor) using a 475/28 bandpass filter and a 470/40
excitation filter in conjunction with a multispectral dichroic (59022 bs,
Chroma). GCaMP6s emission was acquired using a 525/50 filter (Chroma) and
mBeRFP emission was acquired using a 632/60 emission filter (Chroma).

Isolated membrane proteins were diluted to 1–2 mg/mL in
binding buffer (BB) (150 mM NaCl, 20 mM HEPES pH 7.4, 2 mM CaCl₂,
2 mM MgCl₂, and EDTA-free protease inhibitor cocktail) and then
incubated with 1–50 µM of mipATP in a 96-well plate for
25–30 min at 4°C in the dark. For competition experiments,
isolated membranes were preincubated with the indicated competitor compound
(ATP, Sigma #A26209; ATPγS, Tocris #4080; ADP, Sigma #A2754; AMP,
Sigma #A1752; Adenosine, Sigma #A9251; GTP, Sigma #G8877; CTP, Sigma #C1506;
UTP, Sigma #U6750; molybdate, Sigma #243655) for 10 min at 4°C in the
dark prior to the addition of mipATP. Plates containing the samples were
then placed on ice and UV-irradiated (365 nm) (Analytik Jena US;
#95–0045-04) for 15 min at a distance of 2 cm from the light source.
Samples not exposed to UV were placed on a separate 96-well plate that was
left in the dark at 4°C for 15 minutes instead of UV-irradiated.
Next, membranes were collected and resuspended in 5 volumes of ice-cold BB
and centrifuged at 230,000 × g for 10 min at
4°C. Supernatant was discarded, and the pellets were resuspended in
BB and re-pelleted by centrifugation. Membrane pellets were snap frozen with
liquid nitrogen or solubilized in SDS lysis buffer (4% SDS, 150 mM NaCl, 50
mM triethanolamine (TEA) pH 7.4, EDTA-free protease inhibitor cocktail) for
downstream click chemistry reactions.