An adaptation of published assay (Boncompain et al., 2012) was used. HeLa cells were transfected with Str-KDEL-TNF-SBP-mCherry construct as described above, and 24 h after transfection mCherry positive cells were sorted. 5x10 4 cells were cultured on 35 mm imaging dish. The day after, cells were transferred at 37 o C in a thermostat-controlled chamber. At time point zero, the medium was removed and replaced with medium containing D-biotin (Sigma-Aldrich) at 40 µM concentration.
The time-lapse acquisition was made using a Zeiss LSM800 AiryScan Elyra S1 SR confocal microscope. Images were acquired using a 63x oil-objective. For each time point, the integrated intensity of a region of interest (ROI) was measured. The integrated intensity of an identical size ROI corresponding to background was measured and subtracted from the values of the integrated intensity for each time point.
The values were then normalized to the maximum value. These quantifications were performed using the Zeiss Black software. and stained with mouse-anti-beta-catenin antibody as described above. The acquisition was made using a Zeiss LSM800 AiryScan Elyra S1 SR confocal microscope. Images were acquired using a 40x oil-objective.
The time-lapse acquisition was made using a Zeiss LSM800 AiryScan Elyra S1 SR confocal microscope. Images were acquired using a 63x oil-objective. For each time point, the integrated intensity of a region of interest (ROI) was measured. The integrated intensity of an identical size ROI corresponding to background was measured and subtracted from the values of the integrated intensity for each time point.
The values were then normalized to the maximum value. These quantifications were performed using the Zeiss Black software. and stained with mouse-anti-beta-catenin antibody as described above. The acquisition was made using a Zeiss LSM800 AiryScan Elyra S1 SR confocal microscope. Images were acquired using a 40x oil-objective.