Streptavidin Bead Enrichment of Biotinylated Proteins.

APEX-labeled cell pellets from a T150 flask were used as input for each sample. The pellets were then lysed in RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 1% Triton X-100, protease mixture [Sigma-Aldrich], and 1 mM phenylmethylsulfonyl fluoride) for 5 min at 4 °C. The lysates were cleared by centrifugation at 15,000 × g for 10 min at 4 °C. Streptavidin-coated magnetic beads (Pierce) were washed twice with RIPA buffer, and 8 mg of total protein cell lysate from each sample was separately incubated with 450 µL of magnetic bead slurry with rotation for 1 h at room temperature or overnight at 4 °C. The beads were subsequently washed twice with 1 mL of RIPA lysis buffer, once with 1 mL of 1 M KCl, once with 1 mL of 0.1 M Na₂CO₃, once with 1 mL of 2 M urea in 10 mM Tris⋅HCl (pH 8.0), and twice with 1 mL RIPA lysis buffer. For streptavidin blot analysis, biotinylated proteins were then eluted by boiling the beads in 75 µL of 3× protein loading buffer supplemented with 20 mM dithiothreitol (DTT) and 2 mM biotin, and run on SDS-polyacrylamide gel electrophorsesis (PAGE) gel.