Rabbit anti cd97

Western blot analysis was performed using standard protocols as described previously (26 (link), 27 (link)). All protein extracts were denatured in a boiling water bath (95°C) for 5 min except for NIS, which was heated at 37°C for 30 min. The antibodies and their dilutions used were as follows: rabbit anti-NIS (1:200; Abcam, UK); rabbit anti-CD97 (1:1,000; Abcam, UK); rabbit anti-GAPDH (1:5,000; Proteintech, China); rabbit anti-Na+/K+-ATPase (1:1,000; Abcam, UK); rabbit anti-cAMP (1:1,000; Abcam, UK); rabbit anti-pCREB (Ser133) (1:1,000; Abcam, UK); mouse anti-TSHR (1:1,000; Abcam, UK); peroxidase conjugated goat anti-rabbit IgG (1:10,000; ZSGB-BIO, China); peroxidase conjugated goat anti-mouse IgG (1:10,000; Proteintech, USA). The quantitative analysis of band intensity was performed by ImageJ software.

The cells were collected and subsequently lysed with lysis buffer (CST, USA) to extract total proteins. The 40 μg of proteins was loaded onto a 10% polyacrylamide-SDS gel for separation. Next, the proteins were transferred onto PVDF membrane (Millipore, USA) and the membrane was then blocked by incubating with a 5% nonfat milk for 1 hour at room temperature. Later, membrane was hybridized with primary antibody at 4°C overnight followed by washing with TBST for a total of 30 min, three times. Subsequently, membrane was incubated with corresponding secondary antibody for 1 hour at room temperature. Finally, chemiluminescence was detected using ECL kit (Pierce, USA). The antibodies and their dilutions used were as follows: rabbit anti-CD97 (1 : 1000) (Abcam, USA); rabbit anti-GAPDH (1 : 1000) (Nuoyang, China); mouse anti-p65 (1 : 1000) (CST, USA); rabbit anti-PPAR-γ (1 : 1000) (CST, USA); rabbit anti-Lamin B (1 : 1000) (Nuoyang, China); goat anti-rabbit (1 : 5000) (Nuoyang, China); goat anti-mouse (1 : 5000) (Nuoyang, China).