Anti er f 10

After washing beads as described in ‘BAR’, biotinylated proteins were eluted incubating beads in SDS protein loading buffer with 2 mM biotin and 20 mM dithiothreitol for 10 min at 95 °C.
For total protein extraction, cells were lysed in extraction buffer (1% (v/v) Triton X-100, 10 mM Tris-HCl pH 7.4, 5 mM EDTA, 50 mM NaCl, 50 mM sodium fluoride, 2 mM Na₃VO₄, Complete inhibitor mix (Roche Applied Science) and homogenized by passage through a 26-gauge needle six times. The lysate was incubated on ice for 5 min and then clarified by centrifugation (14,000 rpm for 10 min at 4 °C). The protein concentration was then quantified with Bradford assay (Bio-Rad). Lysates were resolved by SDS-PAGE (Invitrogen) and transferred onto a nitrocellulose membrane (Amersham) for immunoblot analysis. After 1 h blocking with 5% milk in TBST at RT for 1 h, membranes were incubated with primary antibodies overnight at 4 °C. Following antibodies were used: anti-ER F10 (#sc-8002 Santa Cruz Biotechnology), streptavidin HRP conjugate (#S911 Thermo Scientific) anti-Flag (#F1804 Sigma-Aldrich) and tubulin (#T9026 Sigma).