Phosphorylated cofilin

Total cell lysates were isolated using lysis buffer (Tris 25 mM, sodium chloride 150 mM, 0.1% Sodium dodecyl sulphate, 0.5% sodium deoxycholate, 1% Triton X100). Western blotting was carried out using specific antibodies against phosphorylated cofilin (Cell Signalling Technology), phosphorylated MLC (Cell Signalling Technology), and pyruvate dehydrogenase complex component X (PDHX; Cell Signalling Technology) with horse radish peroxidase-conjugated secondary antibodies (Dako). Chemiluminescent detection was carried out using ECL Prime^® (GE Healthcare). The protein levels of tissue factor pathway inhibitor (TFPI) or von Willebrand factor (vWF) in cell lysates or cell culture supernatants were quantified by enzyme-linked immunosorbent assay (ELISA; AbCam).

Cell extracts were separated on 12% SDS-PAGE gels and transferred to nitrocellulose membranes, which were blocked with 5% milk powder in Tris-buffered saline (10 mM Tris-HCl, pH 8, 150 mM NaCl). The membranes were incubated overnight at 4°C with mouse anti-Wdr1 (Santa Cruz, #sc-393159; 1:500), sheep anti-Cotl1 (R&D Systems, #AF7865; 1:500), rabbit anti-CD79a (1:5,000) (Gold et al., 1991 (link)), or the following rabbit antibodies from Cell Signaling Technologies: phosphorylated CD79a (pCD79a; #5173; 1:1,000), phosphorylated Erk (pERK; #9101; 1:1,000), Erk (#9102, 1:1,000), phosphorylated cofilin (p-cofilin; #3313; 1:1,000), or cofilin (#3318; 1:1,000). Immunoreactive bands were visualized using horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad, #170-6515; 1:3,000), mouse Igκ-binding protein (Santa Cruz, #sc-516102, 1:2,000), or donkey anti-sheep IgG (R&D Systems, #HAF016, 1:1,000), followed by ECL detection (Azure Biosystems, #AC2010). All antibodies were diluted in Tris-buffered saline. Blots were quantified and imaged using a Li-Cor C-DiGit imaging system.