Adcy1

Protein expression levels of candidate targets of HYD or the herb pair HZ-GC in thyroid tissues obtained from different groups were detected by Western blotting analysis as described in our previous study [41 (link)]. Antibodies against the following proteins were used: Adcy1 (rabbit polyclonal antibody, dilution 1:200, Abcam, Cambridge, UK), Adcy2 (rabbit polyclonal antibody, dilution 1:200, Abcam, Cambridge, UK), Atp1a2 (rabbit monoclonal antibody, dilution 1:500, Abcam, Cambridge, UK), Creb1 (rabbit monoclonal antibody, dilution 1:500, Abcam, Cambridge, UK), Gsr (rabbit monoclonal antibody, dilution 1:2000, Abcam, Cambridge, UK), Hspa5 (rabbit monoclonal antibody, dilution 1:2000, Abcam, Cambridge, UK), Lyd (rabbit polyclonal antibody, dilution 1:1000, Abcam, Cambridge, UK), Pdia4 (rabbit polyclonal antibody, dilution 1:1000, Abcam, Cambridge, UK), Plcb1 (rabbit monoclonal antibody, dilution 1:1000, Abcam, Cambridge, UK), Prkca (rabbit monoclonal antibody, dilution 1:2000, Abcam, Cambridge, UK), Prkcb (rabbit monoclonal antibody, dilution 1:2000, Abcam, Cambridge, UK), Tg (rabbit monoclonal antibody, dilution 1:40000, Abcam, Cambridge, UK), and Tpo (goat polyclonal antibody, dilution 1:500, RD, Minnesota, US). All experiments were performed in triplicate. The mean normalized protein expression ± S.D. was calculated from three independent experiments.

Total protein content was obtained from the lysed cells using a radio-immunoprecipitation assay (RIPA) kits (R0010, Solarbio, Beijing, China) and then quantified using a bicinchoninic acid (BCA) quantitative kit (G3522-1, Guangzhou Jabes Biotechnology Co., Ltd., Guangzhou, China). Next, the protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane. After being blocked 5% bovine serum albumin (BSA) at room temperature for 1 h, the membrane was incubated with diluted primary antibodies purchased from Abcam, Cambridge, MA, USA: ADCY1 (1 : 10000, ab203204), CD133 (1 : 1000, ab216323), CD44 (1 : 1000, ab157107), leucine-rich G protein-coupled receptor-5 (LGR5) (1 : 1000, ab75732), and aldehyde dehydrogenase isoform 1 (ALDH1) (1 : 500, ab129815) overnight at 4° C. Next, the membrane was incubated with the horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (1: 5000, Beijing Zhongshan Biotechnology Co. Ltd., Beijing, China). Finally, an enhanced chemiluminescence (ECL) reagent kit was employed to visualize the results after which the gray value was calculated, with GAPDH regarded as the internal control. The cellular experiment was repeated three times to obtain the mean value.