Protein Extraction and Western Blot Analysis

Protein lysates were added into the tissues after rinsing with pre-cooled PBS for 3 times, lysed at 4 °C and centrifuged (10,000 r/min) for 15 min. Supernatant proteins were then extracted and mixed with sodium dodecyl-sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) loading buffer. Primary antibodies were put into them after SDS-PAGE gel electrophoresis and transfer to a membrane, and the proteins were placed overnight at 4 °C. Then horseradish peroxidase-conjugated secondary antibodies were incubated with the proteins at room temperature. In the end, immunoreactive proteins were tested with a chemiluminescent enhanced chemiluminescence assay kit (GE Healthcare, Uppsala, Sweden) and observed with a LAS3000 luminescent image analyzer (Fujifilm, Tokyo, Japan) with β-actin as internal reference [39 (link)].