Ab85052

Cell lysates were harvested and quantified via BCA Protein Assay Kit (Thermo Fisher Scientific, USA). Secondary antibodies were labeled with HRP (Sigma-Aldrich, USA) and the signals were detected using ECL Kit (Millipore, USA). A β-Actin served as an internal control for the whole-cell lysates. Antibody against NR4A1 (ab109180, dilution 1:1000) and LEF1 (ab85052, dilution 1:1000) were purchased from Abcam (USA), β-actin (A1978, dilution 1:10000) were purchased from Sigma-Aldrich (USA).

Tail tissues (3 mm) before the injury site were excised and frozen in liquid nitrogen and stored at -80 °C. Protein samples were extracted and adjusted to identical concentration. Protein mixtures were loaded onto different percentage SDS-polyacrylamide gels (Bio-Rad) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). Primary antibodies used were as follows: Akt (1:1000, 4685, Cell Signaling Technology (CST)), p-Akt Ser473 (1:1000, 4060, CST), p-Akt Thr308 (1:1000, 13038 S, CST), AMPKα (1:1000, 5832, CST), p-AMPKα Thr172 (1:1000, 2535, CST), mTOR (1:1000, 2983, CST), p-mTOR (1:1000, 2976, CST, 2976), p-p70 S6K Thr389 (1:1000, 9205, CST), p70 S6K (1:1000, 2708, CST), LEF1 (1:1000, ab85052, Abcam), β-catenin (1:1000, ab32572, Abcam), WE6 (1:5, WE6-s, Developmental Studies Hybridoma Bank) and β-actin (1:2000, ab8826, Abcam). β-actin was used as an internal control.