Inhibitor cocktails

N2a cells overexpressing EGFP-fused target molecules were lysed in 1 ml buffer containing 50 mM Tris.HCI pH 7.4, 150 mM NaCl, 0.5% Triton X-100, inhibitor cocktails (Roche), 1 mM PMSF, and 100U/ml Turbonuclease (Sigma). Lysates were cleared by centrifugation at 17,000 g for 15 min at 4°C and incubated with 12 μl GFP-Trap magnetic beads (Chromotek) for 1 h at 4°C with gentle rotation. Beads were washed three times with 1 ml buffer containing 50 mM Tris.HCI pH 7.4, 300 mM NaCI, 0.5% Triton X-100, 1 mM PMSF. Bead-bound proteins were denatured by boiling in 15 μl sample buffer before SDS-PAGE. Ten μl of the boiled-sample was loaded for probing with target antibodies. Five μl of the samples was used to probe with GFP antibody as expression control. Total protein lysate and IP fractions were analyzed by immunoblotting using primary antibodies against β-Actin (sc-47778, SCBT), Profilin-2 (sc-100955, SCBT), GFP (GFP-1020, AvesLab), FLAG M2 (F3165, Sigma) and SHTN1 (kindly provided by Dr. O. Reiner) [46 (link)]. Primary antibodies were detected by appropriate fluorophore-conjugated secondary antibodies (donkey anti-mouse IgG Alexa Fluor 647, donkey anti-rabbit IgG Alexa Fluor 568 or goat anti-chicken IgG Alexa Fluor 488; Thermo). Blots were visualized by Typhoon FLA9000.