Ab215711

The African green monkey kidney epithelial cells Vero 81, human cervical cancer cell line HeLa, and human hepatoma cell line Huh-7 were maintained in DMEM medium supplemented with 10% FBS. The human lung adenocarcinoma cell line Calu-3 and colorectal adenocarcinoma cell line Caco-2 were cultured in Minimum Essential Medium (MEM, Gibco, catalog 11095080) containing 10% and 20% FBS, respectively, supplemented with 1% Non-Essential Amino Acids Solution (Gibco, catalog 11140050) and 1 mM Sodium Pyruvate (Gibco, catalog 11360070). The bat lung cell line Tb 1 Lu were maintained in MEM medium supplemented with 10% FBS. The human bronchogenic carcinoma cell line ChaGo-K-1 was grown in Roswell Park Memorial Institute 1640 Medium (RPMI 1640, Gibco, catalog C11875500BT) containing 10% FBS. The human bronchial epithelial cell line BEAS-2B was incubated in Bronchial Epithelial Cell Growth Medium BulletKit (BEGM, LONZA, catalog CC-3170). All cells were grown at 37 °C in a humidified 5% CO₂ atmosphere. The hDPP4 and hACE2 expression in different cell lines was determined by western blotting assay using Anti-DPP4 antibody (Abcam, ab215711, 1:1000 dilution), Anti-ACE2 antibody (Abcam, ab108209, 1:1000 dilution), HRP-labeled Goat Anti-Rabbit IgG(H + L) (Beyotime, A0208, 1:1000 dilution).

Thyroid tissue was homogenized in lysate supplemented with protease inhibitors and phosphatase inhibitors, and total protein was collected after centrifugation. Protein concentration was measured with the BCA protein assay kit (Thermo Scientific). Tissue lysates were mixed with sodium dodecyl sulfate sample buffer, boiled, and separated by 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The proteins were then electrotransferred to polyvinylidene difluoride (PVDF) membranes, which were incubated overnight at 4 °C with anti-DPP4 antibody (ab215711, Abcam, RRID: AB_2734752). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (10494-1-AP, Proteintech, RRID: AB_2263076) was used as control. PVDF membranes were then incubated with peroxidase-conjugated secondary antibodies, and specific bands were detected with a Bio-Rad imaging system. Band intensity was measured using ImageJ software.