Electrogenerated chemiluminescence system

The tissues or cells were homogenized in lysis buffer (50 mM Tris, pH 7.4, 150 mMNaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS) containing protease inhibitors (Roche Applied Science). The lysates were clarified by centrifugation at 13,000 × g at 4 °C for 20 min, and the supernatants were collected and normalized for protein concentration. Proteins were separated by 8% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Immobilon-P, Millipore). After blocking with PBS containing 5% skim milk and 0.05% Tween 20, the membranes were incubated with primary antibodies.For detection, a fluorescence-conjugated secondary antibody and an electrogenerated chemiluminescence system (GE Healthcare) were used. The membrane was exposed to an imaging system (LAS-3000, Fujifilm) according to the manufacturer’s specifications. The protein bands were quantified using ImageJ 1.44p software. The following antibodies were used: rabbit anti-p-ERK1/2 (1:1000,Santa Cruz, sc-7383); rabbit anti-ERK1/2 (1:1000,Santa Cruz,sc-292838), rabbit anti-GAPDH (1:5000, Abcam, ab128915). Horseradish peroxidase-conjugated rabbit IgG-specific (1:5000, Cell Signaling Technology, 7074 S) were used for secondary antibodies. Full-length gel is available at Supplementary data.

Total protein extraction from tissues and cell lines was implemented in Radio-Immunoprecipitation assay reagent (Beyotime Biotechnology Co., Ltd., Shanghai, China) with protease inhibitor phenylmethylsulfonyl fluoride (Sigma-Aldrich). The protein sample (20 μg) at the same amount was separated via sulfate polyacrylamide gel electrophoresis, and transferred to Polyvinylidene fluoride films (Millipore Corporation, Billerica, Massachusetts, USA) [18 (link)]. After blocking in 5% skimmed milk, the films were received with incubation with the following primary antibodies (Abcam Inc., Cambridge, MA, USA): MAPRE1 (ab50188), GAPDH (ab8245) at 4°C overnight. After wash with phosphate buffer saline (PBS) buffer for 3 times at room temperature, the films were received with incubation with secondary antibody (Abcam) for 120 minutes. At last, the protein bands were received with visualization on electrogenerated chemiluminescence system (GE Healthcare Bio-science, U.S.) and quantification was via Image J software with GAPDH as an internal reference.