Immunofluorescence and live-cell Imaging

U2OS cells were washed once with PBS and immediately fixed by 4% EM-grade paraformaldehyde for 0h 10m 0s at Room temperature

Cells were washed three times with PBS for 0h 10m 0s each time.

Blocked and permeabilized for 0h 30m 0s in permeabilization buffer (5% FBS and 0.1% saponin in PBS)

Cells were then incubated with 1:100 dilution of primary antibodies at 4°C 1h 0m 0s

Cells were washed three times with PBS for 0h 10m 0s each time.

Cells were incubated with 1:500 dilution of fluorophore-conjugated secondary for 0h 30m 0s at Room temperature

Prolong Gold with DAPI was used as mounting solution

Images were acquired with a Zeiss LSM900 confocal microscope and analyzed with Fiji/ImageJ software

Cells were fixed with 4% paraformaldehyde in PBS and 0.1% Triton-X was used for permablization 0h 10m 0s

Blocked in 10% normal donkey serum for 1h 0m 0s Room temperature

Cells were then incubated with 1:100 dilution of primary antibodies at 4°C 1h 0m 0s

Cells were washed three times with PBS for 0h 10m 0s each time.

Cells were incubated with 1:500 dilution of fluorophore-conjugated secondary for 0h 30m 0s at Room temperature

Prolong Gold with DAPI was used as mounting solution

Images were acquired with a Zeiss LSM900 confocal microscope and analyzed with Fiji/ImageJ software

Cells were cultured in 35 mm glass bottom dishes (MatTek).

HaloTag fluorescent ligands were added according to the manufacturer’s protocol (Promega).

After incubation for 0h 15m 0s in the incubator (37°C and 5% CO2 ), the cells were quickly washed twice with PBS. The medium was replaced with Opti-MEM supplemented with 10% FBS.

Imaging was performed using a Zeiss LSM900 confocal microscope in a temperature-controlled

(37°C and 5% CO2) environment.