FRAP experiments were performed on an Olympus FV3000 confocal microscope system, similar to previous studies66 (link),74 (link). For in vitro protein droplets, droplets of ~2 µm diameter were photobleached using 488 nm and 546 nm lasers at around 10% laser power and maintained for 4 s. After bleaching, time-lapse images were obtained at ~2 min intervals of about 1 s.
For cellular droplets, FRAP experiments were performed in a live-cell imaging chamber at 37 °C. HeLa cells were transfected with the pEGFP-TRIM25 plasmid for 6 h and then treated with poly(I:C) for 6 h. Complete or partial photobleaching of the target droplets was achieved using a laser at 488 nm with about 10% laser power for 4 s. After bleaching, time-lapse images were obtained over a time course of 1.5 min.
The fluorescence intensity of the region of interest (ROI) above was corrected by the unbleached control region and then normalized to the pre-bleached intensity of the ROI. The corrected and normalized data were fitted to a single exponential model using the OlyVIA software developed by Olympus.
For cellular droplets, FRAP experiments were performed in a live-cell imaging chamber at 37 °C. HeLa cells were transfected with the pEGFP-TRIM25 plasmid for 6 h and then treated with poly(I:C) for 6 h. Complete or partial photobleaching of the target droplets was achieved using a laser at 488 nm with about 10% laser power for 4 s. After bleaching, time-lapse images were obtained over a time course of 1.5 min.
The fluorescence intensity of the region of interest (ROI) above was corrected by the unbleached control region and then normalized to the pre-bleached intensity of the ROI. The corrected and normalized data were fitted to a single exponential model using the OlyVIA software developed by Olympus.