Ab168377

Denaturing Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) sample buffer was used to dissolve the cells by the standard method. Protein lysates of ox-LDL (50 μg/ml) induced HUVEC cells (for 24 h) were extracted using RIPA lysis buffer supplemented with cocktail of protease inhibitors (Roche Diagnostics, Indianapolis, IN) then were transferred onto nitrocellulose membranes after 10% SDS-PAGE was used to separate them. Then the Tris Buffered Saline (TBS) which contained 0.1% Triton X-100 and 5% nonfat milk was added into the membranes and blockaded at the temperature of 4°C after that. Overnight passed, the primary first antibodies, including anti-LDH (ab222910, 1/1,000; Abcam, Cambridge, MA), anti-IL-6 (ab6672, 1/1,000; Abcam), anti-IL-1β (AB10626, 1/1,000; Merck Millipore, Darmstadt, Germany), anti-GNAI2 (ab157204, 1/10,000; Abcam), anti-PCBP1 (ab168377, 1/10,000; Abcam) and loading control anti-GAPDH (ab8245, 1/10,000; Abcam) were used to incubate the membranes. This process still took one night. Then the membranes were incubated with the HRP-labeled secondary antibodies (Abcam) for 2 h at room temperature. The signals were detected on an ECL system (Amersham Pharmacia, Piscataway, New Jersey). GAPDH served as endogenous control.