Anti flag m2 antibody beads

Pulse-chase experiments were performed as described previously [13 (link)]. In brief, cells were labeled for 30 min at 37 °C in cysteine/methionine-free DMEM supplemented with 5% dialyzed FBS and 0.1 mCi/mL (³⁵S)cysteine/(³⁵S)methionine (PerkinElmer, Waltham, MA, USA). After washing with ice-cold PBS, cells were chased in normal culture medium at 37 °C. Cells were lysed in TNE buffer and then subjected to immunoprecipitation with anti-FLAG M2 antibody beads (Sigma-Aldrich). Immunoprecipitates were analyzed by SDS–PAGE and autoradiography using a FLA7000 phosphorimager (Fujifilm, Tokyo, Japan).

HeLa cells were transfected with constructs encoding selected proteins, along with the construct encoding tagged HAX1. Cells were harvested for immunoprecipitation 24 h following transfection, lysed in NP-40 lysis buffer (10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% Nonidet P40) supplemented with Complete Protease Inhibitor Cocktail (Roche, 05056489001). After preincubation with protein A-coated agarose beads (Dynabeads, Invitrogen), equal amounts of protein (200 μg) from cell lysates were immunoprecipited with 40 μl of anti-FLAG M2 antibody beads (Sigma), anti-c-Myc Magnetic Beads (Pierce) or GFP-trap (Chromotek).

HEK293T cells were lysed in ice‐cold lysis buffer (NaCl 150 mM, Tris–HCl 50 mM pH 7.4, EDTA 2 mM) containing 1% Triton X‐100, 0.1% sodium dodecyl sulfate, 10% glycerol, 1 × cOmplete Ultra tablets protease inhibitor cocktail (Roche), 1 mM sodium orthovanadate (Sigma‐Aldrich), 11 mM β‐glycerolphosphate, and 10 mM sodium fluoride (Sigma‐Aldrich). Each cell culture dish containing 3 × 10⁶ cells was treated with 1 ml lysis buffer with or without 2 μM rapamycin on ice, collected in a centrifuge tube, and spun down at 14,000 × g, 4°C. For each cell culture dish, 100 μl of supernatant were used as input and diluted with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE) sample buffer, and 900 μl of supernatant were used for immunoprecipitation with 10 μl of the anti‐Flag M2 antibody beads (Sigma‐Aldrich) for 2–4 h at 4°C. Beads were washed with 1 ml of lysis buffer three times. Bound proteins were eluted with SDS‐PAGE sample buffer or Flag‐peptide (Sigma‐Aldrich). Input and immunoprecipitated proteins were boiled for 5 min, and subjected to western blotting. Spodoptera frugiperda, Sf21 (baculovirus)‐derived BDNF protein (Bio‐Techne) was used to compare the biological activity of BDNF obtained from pro‐TEVcs‐BDNF.