Prkca

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.

Protein extracts were resolved by SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were probed with primary antibodies, and then with peroxidase-conjugated secondary antibody (Beyotime, Suzhou, China), followed by visualization using chemiluminescence assay (Pierce, Rockford, IL, USA). Antibodies against EIF4B, FOXO4, PRKCA (Abcam, Cambridge, UK), TET3 (Abnova, Taipei, Taiwan), cleaved caspase-3 (Asp175; Cell Signaling Technology, Danvers, MA, USA), and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used in this study.