BioID proximity biotinylation

To test whether the TMEM16K BioID-fusion proteins were enzymatically active and capable of biotinylating, HEK293 cells were transfected with myc-BioID-TMEM16K or TMEM16K-BioID-HA. Biotin was added (50 µM) for 1 h or overnight, cells were fixed, and biotinylation was visualized with a streptavidin probe conjugated to a fluorescent label.

To purify protein complexes surrounding TMEM16K via in situ BioID-catalyzed biotin labeling, we transfected HEK293 cells with BioID-myc-TMEM16K or TMEM16K-HA-BioID and incubated with 50 µM biotin overnight (12–16 h). Mock transfected cells were processed in parallel to account for endogenously biotinylated proteins. Cells were washed 3× with ice cold PBS and lysed with lysis buffer with protease inhibitors (Roche). Cell lysate was centrifuged for 30 min at 15,000 g and supernatant was applied to streptavidin-conjugated magnetic beads (Dynabeads^® MyOne™ Streptavidin C1, Invitrogen, # 65001). Beads were extensively washed 5× with lysis buffer followed by 5× washes with ice-cold PBS, and flash frozen in liquid nitrogen and stored at −80 °C for mass spectrometry. The experiment was performed in a minimum of three biological replicates per conditions.