proximity labeling 4T1 Cells through PCN

The 4T1 cells were labeled with BxxP via HA-PCN nanozyme as described above. After washing 5 times with PBS, cells were collected and lysed in RIPA buffer containing 1 % (v/v) Protease inhibitor cocktail (ThermoFisher Scientific) and 1 % (v/v) PMSF under bath-sonication, followed by centrifugation for 10 minutes at 4 ℃ to remove cell debris. Before the enrichment experiment, Pierce streptavidin magnetic beads (ThermoFisher Scientific) were washed twice with RIPA buffer for 2 minutes. The 400 μg samples were mixed with 50 μL beads slurry with rotation for 2 hours at 4 ℃, followed by washing twice with 1 mL RIPA buffer for 2 minutes, once with 1 mL 1 M KCl for 2 minutes, once with 1 mL 0.1 M Na(2)CO(3) for 2 minutes, once with 1 mL 2 M urea (pH=7.5) for 2 minutes, twice with RIPA buffer for 2 minutes []. Before enrichment, 100 μL cell lysis was packed as the input sample. For the western blotting analysis, the labeled proteins were eluted from the beads by boiling with protein loading buffer containing 20 mM dithiothreitol (DTT, ThermoFisher Scientific) and 2 mM biotin.