β actin

The total protein was obtained from leaf ventricular myocardial tissues after extraction with the RIPA lysis buffer (P0013B, Beyotime, Chine). Protein concentration was measured by the Pierce™ BCA Protein Assay Kit (Thermo Scientific, USA). A total of 60 μg total protein was electrophoresed and separated with 10% SDS-PAGE and transferred onto nitrocellulose membranes (Millipore, US). After blocking with 5% fat-free milk at room temperature for 2 h, the membrane was incubated overnight at 4°C with primary antibody raised in mice against Akt (1 : 1000), FOXO3 (1 : 1000), Beclin (1 : 1000), Actin (1 : 1000), pi3k (1 : 1000), mTOR (1 : 1000), p-Akt (Ser473, 1 : 1000), GSK-3β (1 : 1000), and TORC2 (1 : 1000). All antibodies were purchased from Cell Signaling Technology (USA). Then, the membranes were incubated with HRP-conjugated secondary antibodies (1 : 2000; Cell Signaling Technology, USA) at room temperature for two hours. The immunoreactive bands were visualized using the enhanced chemiluminescence kit (ECL Millipore Corp., Bedford, MA, USA) in a western blotting detection system (Bio-Rad, CA, USA). Developed films were scanned and Image-ProPlus 5.1 was used for quantitative analysis. In this experiment, β-actin (1 : 1500, Bioss, Beijing, China) was used as the standard and the results were expressed as density values normalized to β-actin.

All protein samples were prepared as follows: 0.5 g of tissue was frozen in liquid nitrogen, ground to yield tissue powder, and then suspended in ice-cold RIPA lysis buffer (ABcom Co., Ltd., Shanghai, China). The suspension was then sonicated for 1 min at 0–8°C and centrifuged at 8000 ×g for 30 min. The protein concentration was determined by BCA (Sigma, Shanghai, China) assay. The lysates were then separated electrophoretically in 12% polyacrylamide gels and transferred onto PVDF membranes (Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China). The membranes were blocked for 1.5 h at room temperature in 5% nonfat milk and incubated overnight at 4–8°C with TSHR, TTF-1, and PAX8 antibodies (the same as those used in immunohistochemistry), respectively. After the triple wash in Tris buffered saline with tween (TBST) for 30 min, the membranes were incubated with HRP-conjugated secondary antibodies for 45 min and then triple washed again in TBST. Immune reactive bands were revealed using ECL detection system. In the negative group, 2% BSA was used instead of the primary antibodies. β-actin (Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China) was also detected as an internal control. The X-ray film was scanned, and the band density was calculated using ImageJ 1.45 software.

Protein concentration of each liver sample was determined as previously described60 (link). The protocol for immunoblot analysis was also previously described60 (link). The following antibodies were used in this study: Complement C5 (Cat. No. bs-15197R, Beijing Biosynthesis Biotechnology Co., Ltd. Beijing, China), TNFα (Cat. No. bs-10802R, Beijing Biosynthesis Biotechnology Co., Ltd. Beijing, China) and β-Actin (Cat. No. sc-47778; Santa Cruz Biotechnology, Inc., Santa Cruz, CA).