Genegnome chemiluminescent imaging system

Cells were lysed in protein lysis (1% deoxycholic acid, 10 mM Na4P₂O₇, 1% TritonX-100, 10% glycerol, 100 mM NaCl, 5 mM EDTA (pH 8.0), 20 mM Tris-HCl (pH 7.4), 0.1% SDS, 50 mM NaF, 1 mM Na₃VO₄, 1 mM PMSF, 10 mg/L aprotinin). Equal amounts of protein were separated on SDS-PAGE gels and transferred to nitrocellulose membranes. The following primary antibodies were used: anti-phospho-ERK1/2 (no. 4695, Cell Signaling), anti-ERK (no. 9102, Cell Signaling), anti-GAPDH (no. 2118, Cell Signaling), anti-phospho-Src^Y416 (no. 2101, Cell Signaling), anti-Src (no. 2109, Cell Signaling), anti-flag (no. F9291, Sigma), anti-PCNA (no. sc-56, Santa), anti-α-SMA (no. ab5649, Abcam). After treated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (Zhongshanjinqiao, China), the protein bands were detected using an enhanced chemiluminescence System (Millipore, U.S.A.) and visualized on a GeneGnome chemiluminescent imaging system (Syngene, U.K.).

Cells and heart tissue were lysed in protein lysis (1% Deoxycholic acid, 10 mM Na₄P₂O₇, 1% TritonX-100, 10% Glycerol, 100 mM NaCl, 5 mM EDTA (pH = 8.0), 20 mM Tris-HCl (pH = 7.4), 0.1% SDS, 50 mM NaF, 1 mM Na₃VO₄, 1 mM PMSF, 10 mg/L aprotinin). Equal amounts of protein were separated on 10% SDS-PAGE gels and transferred to nitrocellulose membranes. After blocking with 5% milk for 1 h, the membranes were incubated with primary antibodies at 4 °C overnight and incubated with secondary antibodies for 1 h at room temperature. After being treated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (Abacm, ab97051, Cambridge, MA, USA), the protein bands were detected using a chemiluminescence reagent (Affinity, KF8003, San Francisco, CA, USA) and visualized on a GeneGnome chemiluminescent imaging system (Syngene, Iselin, NJ, USA) [9 (link)]. The relative density of each band was analyzed using ImageJ. β-Tubulin and GAPDH served as internal references in densitometric analysis. The primary antibodies used in this study are given in Table 2.

The total proteins of PC12 cells and rat hippocampi were extracted using RIPA lysis buffer. The total protein concentration in samples was measured by a BCA assay kit. The protein samples were separated on sodium dodecyl sulfate-polyacrylamide gels and transferred from the gels onto polyvinylidene fluoride membranes (Roche Applied Science, Indianapolis, IN). After blocking with 5% (w/v) skimmed milk in tris-buffered saline containing 0.1% Tween-20 (TBST) for at least 1 h at RT, the membranes were incubated with appropriate primary antibodies at 4 °C overnight. On the following day, the membranes were washed and incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies for 1 h at RT. After washing three times with TBST, the band intensities were visualized using enhanced chemiluminescence solution, scanned by a GeneGnome chemiluminescent imaging system (Syngene, Cambridge, UK), and quantified with Quantity One software (Bio-Rad, Hercules, CA, USA).