293T cells were transfected with plasmids of mTagBFP-Piezo1 and GFP-TLR4, CFP and YFP, or BFP–GFP fusion protein. FRET analysis was performed according to the acceptor photobleaching method using a confocal microscope ZEISS LSM-780 (Carl Zeiss). Photobleach was done by 488 nm laser irradiation, repeated 200 times. The excitation wavelength was 405 and 488 nm for detecting fluorescent signal of mTagBFP and GFP59,60, respectively. These emissions were collected at 440–480 nm and 500–560 nm for BFP and GFP, respectively. FRET efficiency was calculated according to the equation (ZEN black), FRET efficiency (%) = (D Post − D Pre)/D Post × 100. D Post: BFP signal after photobleaching; D Pre: BFP signal before photobleaching.
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