Anti integrin αvβ3 polyclonal antibody

The expression of integrin αvβ3 was evaluated by western blot and normalized to that of β-actin [34 (link)]. Hth7, 8505C, THJ16T, Cal-62, and Nthy-ori 3-1 cells were plated in 6-well plates and cultured in cell-based medium containing 10% FBS for 24 h. Cells at ~ 80% confluence were used for subsequent protein extraction. The different cells were washed with 1 × PBS 3 times and then lysed in RIPA buffer supplemented with PMSF for 15 min at 4 ℃. Proteins were separated by 10% SDS-PAGE and then transferred to PVDF membranes (Millipore, USA). The immunoblots were blocked with 1 × PBS-5% fat-free dried milk for 1.5 h at room temperature and then incubated at 4 ℃ with anti-integrin αvβ3 polyclonal antibody (1:1000, Bioss, China) and anti-β-actin antibody (1:5000, Abcam, UK) for more than 16 h. After incubation with the HRP-conjugated secondary antibody (1:5000, Bioss, China) for 1 h, the PVDF membranes were visualized with an enhanced chemiluminescence system kit (Millipore, Bedford, MA, USA), and the grayscale of the strip was analyzed by ImageJ [28 , 34 (link), 35 (link)].

Paraffin-embedded tissue samples (including ATC cancerous tissues and the paired adjacent noncancerous tissues) from 8 ATC patients (8 pairs, 16 samples total) were sectioned and stained with a 1:200 dilution of anti-integrin αvβ3 polyclonal antibody (Bioss, China) according to the manufacturer’s protocol. Positive staining was identified with a DAB system (Jinqiao, Zhongshan, China). Six regions were randomly selected for each specimen.