Western Blot Analysis of CRLF1 and Signaling

Medium was collected from cultured BMSCs transduced with empty AAV or AAV containing CRLF1 and combined with 4 x NuPAGE SDS sample buffer without and with reducing agent (Invitrogen, Carlsbad, CA, USA). After denaturation at 70 °C for 10 min, the medium samples were analyzed by electrophoresis in 10% bis-Tris polyacrylamide gels followed by electroblotting onto nitrocellulose filters. After blocking with a solution of low-fat milk protein, blotted proteins were immunostained with primary antibodies specific for β-actin (#3700, Cell Signaling Technology, Danvers, MA, USA), CRLF1 (ab211438, Abcam, Boston, MA, USA) or CLC (ab154798, Abcam), total STAT3 (#9139, Cell Signaling Technology), phosphorylated STAT3 (#9145, Cell Signaling Technology), total Smad2 and 3, phosphorylated Smad 2, and phosphorylated Smad3 (SMAD2/3 Antibody Sampler Kit #12747, Cell Signaling Technology), and then peroxidase-conjugated secondary antibody (Thermo Scientific Pierce, Waltham, MA, USA). The signal was detected by enhanced chemiluminescence (Pierce) as previously described [12 (link)].