Protein Expression Analysis in Mouse Tissues

Mouse tissues were crushed into a fine powder in liquid nitrogen and lysed in RIPA buffer supplemented with complete protease inhibitor (Roche, Switzerland). Protein concentration was determined using the BCA method (KeyGEN Bio TECH Nanjing, China). Western blot analysis was performed as previously described (23 (link)). Aliquots (20 μg) resolved by 8% SDS-PAGE gels electrophoresis, transferred to PVDF membrane (Pall, USA). After blocking with 5% non-fat milk in phosphate buffered saline/Tween-20, the membrane was incubated with antibodies specific to SHP-2 (1:1,000; #3397, Cell Signaling Technology, Boston, USA), CDC25A (1:1,000; #137353, Abcam, UK), mTOR (1:1,000; #2983, Cell Signaling Technology, Boston, USA), IGF1R-β (1:1,000; #9750, Cell Signaling Technology, Boston, USA). The antigen-antibody complexes were detected using an enhanced chemiluminescence (Advansta, Menlo Park, USA). The abundance assessed quantitatively using the Image J softwar.