HEK293T cells were seeded onto glass-bottom culture dishes (MatTek, Ashland, MA) and transiently transfected with the SNAP-Flag-BRK and Halo-SMAD4 constructs. Affinity-tagged proteins were fluorescently labeled during growth, either with Halo-Tag TMRDirect ligand (Promega) or SNAP-Cell 505-Star (NEB) or with both ligands according to the manufacturer’s instructions. Images were taken with a ZEISS LSM 780 confocal microscope with argon laser excitation at 573 to 687 nm for TMRDirect and 499 to 526 nm for SNAP-Cell 505-Star. To limit photobleaching, exposure time and laser power were adjusted to enhance image quality. An alternating excitation mode was adopted to eliminate cross-talk between color channels. HaloTag-SMAD4 or SNAP-Tag BRK-YF were ectopically expressed in HEK293T cells and plated at 20% confluency onto glass-bottom MatTek culture dishes (35 mm, no. 2, 14-mm-diameter glass). To label Halo-SMAD4 proteins, the HaloTag TMRDirect ligand was added in a final concentration of 100 nM and incubated the cells overnight. In addition, the SNAP-Cell 505 ligands were added directly to the cells to label SNAP-Tag BRK-YF in a final concentration of 5 μM and incubated the cells for 1 hour at 37°C in 5% CO2. For colocalization, Halo-SMAD4 and SNAP-Flag-BRK-YF constructs were cotransfected into HEK293T cells, and the cells were labeled as indicated above. The cultured media were replaced with OptiMEM to remove background fluorescence before imaging. Cells were stained with Hoechst dye to mark nuclei for 30 min before imaging.

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