P gap43

Transfected cells were fixed, blocked and hybridized with primary antibodies, including P-GAP43 (Abcam; 1:500 dilution), RANBP10 (Proteintech; 1:500) and β-tubulin (Sigma; 1:1,000 dilution) antibodies. The cells were then reacted with secondary antibodies conjugated with Alexa fluor 488, 594 or 647 (Invitrogen), and cellular nuclear were stained by 1μg/mL Hoechst 33342 (Sigma). Fluorescence images were captured using a DM2500 fluorescent microscope (Leica) for analyses. For 3,3'-Diaminobenzidine (DAB) immunohistochemical staining, brain sections were blocked, incubated with c-Fos (Cell signaling; 1:250) and examined with a Vectastain Elite ABC kit (Vector Laboratories) and DAB (Vector Laboratories). The intensity of c-Fos was quantitated using ImageJ (NIH), and results were subjected to statistical analyses.

Cell and brain samples were lysed using a sonicator (Qsonica), and were subjected to Sodium dodecyl sulfate polyacrylamide gel electrophoresis (Bio-Rad). Separated proteins were transferred onto PVDF membranes (Bio-Rad) for Western blotting, and then hybridized with the primary antibodies, including P-ERK (Cell Signaling; 1:1,000 dilution), T-ERK (Cell Signaling; 1:2,000 dilution), P-GAP43 (Abcam; 1:1,000 dilution), T-GAP43 (Genetex; 1:5,000 dilution), PSD95 (Abcam; 1:5,000 dilution), Synaptophysin (Abcam; 1:5,000 dilution), VAMP1 (Abcam; 1:5,000 dilution), RANBP10 (Proteintech; 1:2,000), Flag M5 (Sigma; 1:2,000 dilution), mEM48 (a gift from Dr. Xiao-Jiang Li, Emory University, USA; 1:50 dilution), MAB2166 (Millipore; 1:2,000 dilution) and γ-tubulin (Sigma; 1:10,000 dilution) antibodies. Protein expression levels were determined by an Amersham ECL kit (PerkinElmer) through an imaging system (ChampGel), and expression profiling was quantitated by an ImageJ system (NIH).