Nonidet p

The 293 T cells were collected after trypsinization and resuspended in mammosphere medium. The cells were then transfected with 100 nmol/liter pAc-GFP-nras^61K or pAc-GFP as control. The liver tissues from the 3, 6, 9 and 12 mpf Tg(fabp10:nras^61K) transgenic zebrafish and WT (12 mpf) were collected respectively, and the livers of four fish were pooled to generate one sample. The samples were lysed with RIPA lysis buffer [20 mM Tris/HCl (pH 7.4), 150 mM NaCl, 0.5% Nonidet P40 and 1 × protease inhibitor cocktail (Roche, Penzber, Germany)], and the lysates were incubated with the suitable antibody including anti-β actin (AB clonal, Wuhan, China, AE012), anti-EGFP (Santa Cruz, CA, SC-47778), anti-MEK1, anti-MEK2, anti-ERK, anti-pMEK1/2, anti-pERK.

Hippocampal neurons from E18.5 Munc13-1/2 DKO at a density of 10.000 / cm² were plated into 6 well plates containing monolayer cultures of astrocytes. Neurons were lysed after 15 DIV at 4°C with 50 mM Tris·HCl, pH 7.9, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 250 μM phenylmethylsulfonyl fluoride, 1% Nonidet P-40, and protease inhibitor cocktail-complete mini (Roche Diagnostics, Berlin, Germany). Lysates were mixed with Laemmli Buffer containing 0.3 mM DTT, and boiled 10 min at 95°C. 30 µg of protein lysates were used for the SDS-PAGE electrophoresis. After separation by SDS-PAGE proteins were transferred to a polyvinyl difluoride (PVDF) membrane. Membranes were blocked with 5% skim milk in TBST, and incubated at 4°C over night with primary antibodies: anti-Flag M2 (F1804; Sigma-Aldrich), and anti-Living Colors GFP (632375; Clontech; Mountain View, CA). Secondary antibodies were horseradish peroxidase-conjugated (Jackson ImmunoResearch; West Grove, PA). The immunoreactive proteins were detected by ECL Plus Western Blotting Detection Reagents (GE Healthcare Biosciences; Pittsburgh, PA) in a Fusion FX7 detection system (Vilber Lourmat, Eberhardzell, Germany).