Characterization of ERM-APEX, APEX2-OMM, and APEX2-NES biotinylation by fluorescence microscopy (related to Figure 1—figure supplement 1)

HEK 293T cells stably expressing ERM-APEX2, APEX2-OMM, APEX2-NES, or no APEX2 construct were grown in SILAC media for 11 days (see ‘SILAC labeling of proteomic samples, biotinylation, and processing for mass spectrometry’ below) and plated on human fibronectin-coated glass coverslips in 48-well plates. The next day, the cells were incubated with 200 µl of 500 µM biotin-phenol in media for 30 min at 37°C, 5% CO₂. Hydrogen peroxide (H₂O₂) was then added to a final concentration of 1 mM for 1 min at room temperature to initiate the biotinylation reaction. The reaction was then quenched by washing three times with ‘quencher solution’ (10 mM sodium ascorbate, 10 mM sodium azide, and 5 mM Trolox in DPBS). The cells were then fixed with 3.7% formaldehyde in DPBS for 15 min at room temperature. After fixation, the cells were washed three times with DPBS. Cold methanol was added to permeabilize the cells, and the samples were placed at −20°C for five minutes. The samples were then washed three times with DPBS. The cells were blocked for 1 hr at 4°C with 3% BSA in DPBS.

The samples were rocked in primary antibody (1:500 mouse anti-Flag for APEX2-OMM and APEX2-NES, 1:1000 mouse anti-V5 for ERM-APEX2) in 3% BSA in DPBS for 1 hr at 4°C. After primary antibody incubation, the samples were washed three times with 0.2% Tween-20 in DPBS. The samples were then rocked in secondary antibody (1:750 anti-mouse AF488) and 1:1000 neutrAvidin-AF647 in 3% BSA in DPBS for 1 hr at 4°C. The neutrAvidin-AF647 conjugate was prepared following Invitrogen’s instructions. Finally, the samples were washed three times with 0.2% Tween-20 in DPBS, maintained in DPBS, and imaged using confocal microscopy.

The data in this figure are representative of three independent experiments with >10 fields of view each.