Western Blot Analysis of Key Signaling Proteins

Western blot analysis was performed as previously described [35 (link)]. 100 ug of protein was separated by 10% SDS-PAGE gel and transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk for 2 h and then were incubated at 4°C overnight with primary antibodies. The primary antibodies for CCR4, p-p65, p65, β-catenin and MMP13 were purchased from Abcam, and antibodies against ERK (4695), p-ERK (4377), JNK (9252), p-JNK (4668), p38 (9212), p-p38 (4631), Akt (4691), p-Akt (13038), and Runx2 (8486) were purchased from Cell Signaling (Danvers, MA). Horseradish peroxidase-conjugated secondary antibodies were used and the protein bands were visualized by an Odyssey scanner (LI-COR Biosciences).