Anti gadph

Cells were lysed using radioimmunoprecipitation assay lysis buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) for 30 min on ice, sonicated for 5–10 sec and then centrifuged at 12,000 × g for 20 min at 4°C. Protein concentrations were determined using a DC^™ Protein Assay (Bio-Rad Laboratories, Inc.). Equal amounts (40 µg) of extracted protein samples were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were blocked with 2% milk in PBS containing 0.1% Tween-20 at room temperature for 2 h, and subsequently probed with the following primary antibodies overnight at 4°C: Anti-SerpinE2 (cat. no. ab154591; 1:1,000; Abcam, Cambridge, MA, USA), anti-tissue plasminogen activator (tPA; cat. no. ab157469; 1:1,000; Abcam), anti-β-actin (cat. no. sc-130300; 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and anti-GADPH (cat. no. ab37168; 1:1,000; Abcam). Membranes were then incubated with the following horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature: Anti-rabbit (cat. no. A0545; 1:5,000; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and anti-mouse (cat. no. A9044; 1:5,000; Sigma-Aldrich; Merck KGaA). The protein bands were detected using an enhanced chemiluminescence detection system (Pierce; Thermo Fisher Scientific, Inc.).