Samples were boiled at 100°C for 5 min with SDS-PAGE loading buffer, then separated on 12% polyacrylamide Tris-glycine gels. Afterwards, the gels were stained with Coomassie Brilliant Blue G-250, and the purity and concentrations of the samples were determined using the Tanon Gis software (Bio-Tanon, China).
For Western blot analysis, protein samples were resolved by electrophoresis and electro-transferred onto PVDF membrane (0.45 μm, Pall, U.S.A.). The membranes were then blocked with 5% fetal bovine serum/Tris-buffered saline-Tween (TBST) overnight at 4°C, after which they were incubated with mouse monoclonal antibody to the P domain of human t-PA (SC-59721, Santa Cruz Biotechnology, U.S.A.) at 37°C for 1.5 h. The membranes were subsequently incubated with HRP-conjugated goat anti-mouse IgG (Sangon Biotech, China) at 37°C for 1 h, followed by three washes in TBST. Immunodetection was conducted using ECL substrate solution (Millipore Corporation, Billerica, MA, U.S.A.) according to the manufacturer’s instructions.
For Western blot analysis, protein samples were resolved by electrophoresis and electro-transferred onto PVDF membrane (0.45 μm, Pall, U.S.A.). The membranes were then blocked with 5% fetal bovine serum/Tris-buffered saline-Tween (TBST) overnight at 4°C, after which they were incubated with mouse monoclonal antibody to the P domain of human t-PA (SC-59721, Santa Cruz Biotechnology, U.S.A.) at 37°C for 1.5 h. The membranes were subsequently incubated with HRP-conjugated goat anti-mouse IgG (Sangon Biotech, China) at 37°C for 1 h, followed by three washes in TBST. Immunodetection was conducted using ECL substrate solution (Millipore Corporation, Billerica, MA, U.S.A.) according to the manufacturer’s instructions.