Phosphorylated erk1 2 p erk1 2

A 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) kit was purchased from Promega (USA). MMP9, ERK1/2, and phosphorylated ERK1/2 (p ERK1/2) antibodies were purchased from Abcam (USA). An RNA extraction kit, reverse transcription kit, and quantitative polymerase chain reaction (qPCR) SuperMIX were purchased from TAKARA (Japan). Puerarin was purchased from Sigma (USA). Matrigel was purchased from BD (USA). The gene amplification instrument and fluorescence imaging system were obtained from Bio-RAD (USA). A microscopic imaging system (Leica, Germany) from Germany was also used.

Cultured BMECs were subjected to the Cell Lysis Buffer System (Santa Cruz, USA) supplemented with phenylmethylsulfonyl fluoride (PMSF, Santa Cruz, USA). A Cytoplasmic Extraction Kit (Beyotime, China) was used to extract the protein according to the manufacturer’s instructions. A BCA kit (Pierce, USA) was used to determine the protein concentrations of the samples, which were then subjected to the vertical SDS-PAGE. The separated proteins were then electronically transferred to PVDF membranes. Primary antibodies against Bax (Abcam, USA), Bcl-2 (Abcam, USA), activated caspase3 (Abcam, USA), p38 (Abcam, USA), phosphorylated p38 (p-p38, Abcam, USA), JNK (Abcam, USA), phosphorylated JNK (p-JNK, Abcam, USA), ERK1/2 (Abcam, USA), phosphorylated ERK1/2 (p-ERK1/2, Abcam, USA), and GAPDH (Abcam, USA) were used to incubate the membranes at 4°C for 8 h. The membranes were washed with TBST and then incubated with HRP-conjugated secondary antibodies. The membranes were developed by using an ECL kit (Pierce, USA). The densities of the immunoblots were determined and analyzed by Gene Genius (Syngene, England) and Image J (VER1.28, NIH, USA). Six independent experiments were carried out for immunoblots density quantification.