Anti rab14

Anti-Rab14 (Sigma-Aldrich) antibody or the control Rabbit IgG was ligated to Dynabeads M450 (Thermo Fisher) according to the manufacturer’s protocol. Briefly, 200 µg goat anti-Rabbit antibody was incubated with 1 mL beads in 1 mL 0.1 M borate buffer for 30 min, then adding 20 µL 5% BSA, rotating at room temperature for 48 h. 200 µL beads were continued to incubate with 10 µg Rabbit Anti-Rab14 antibody or control Rabbit IgG at 4 °C overnight. Endomembrane was prepared as described previously with some modifications⁶⁰ . Briefly, 3 × 10⁸ PLC/PRF/5 cells were washed by pre-cold PBS, scrapped in 1 mL cold PBS, then incubated with hypotonic buffer (10 mM Tris–HCl) for 2 min, the pelleted cells were then resuspended in homogenesis buffer (250 mM sucrose, 3 mM EDTA) with protease inhibitor. Twenty tightly strokes of homogenizer were performed, followed by 3000 × g centrifugation for 10 min. The supernatant was mixed with 62% sucrose to reach a final sucrose concentration of 42%. Discontinuous sucrose gradient ultracentrifugation was conducted, sucrose gradient were 42%, 35%, 25%, 4 mL for every gradient. Then centrifuge at 200,000 × g overnight. Take 1 mL every time from top to bottom. Mix the 35% sucrose part, diluted 5 times with PBS. This fraction was then incubated with the previously prepared beads overnight. For MS/MS detection, beads were eluted by 1% SDS PBS for 5 min.