Anti ph p38

After indicated treatments, RIPA buffer with protease inhibitors (Thermo Fisher Scientific) was added to HUVECs. Thereafter, equal amounts (about 80ug) of total protein samples were separated though 4-20% SDS-PAGE and transferred to PVDF membranes (Merck Millipore) using electroblotting. The membranes were blocked with 5% non-fat milk at the room temperature for 2 h and then incubated overnight at 4 °C with anti-Ph-p38 (1:1000, Cell Signaling Tech #4511), anti-p38 (1:1000, Cell Signaling Tech #9212), anti-Ph-NF-κB p65 (1:1000, Cell Signaling Tech #3033),anti-NF-κB p65 (1:1000, Cell Signaling Tech #8242),anti-VWF (1:1000, Cell Signaling Tech #65707), anti-IL-6 (1:1000, Cell Signaling Tech #12153), anti-TNF-α (1:1000, Cell Signaling Tech #6945),anti-MCP-1 (1:1000, Cell Signaling Tech #81599) and anti-GAPDH (1:1000, Cell Signaling Tech #5174) primary antibodies. The membranes were washed next day and then incubated with HRP-conjugated secondary antibodies (1:1000, Cell Signaling Tech #7074) for 2 h at the room temperature. Finally, an enhanced chemiluminescence system (GE Healthcare) was used to visualize the protein bands. The protein levels were presented as fold change relative to the expression of control. All Western assays were performed in duplicates or triplicates and all experiments were repeated at least three times.