Anti taz antibody

Proteins were obtained using a protein extraction kit (Beyotime, China) according to manufacturers’ instructions. Then, proteins were resolved by 10% SDS/PAGE gel and transferred to 0.22 µm polyvinylidene difluoride (PVDF) membranes (Millipore, U.S.A.). Membranes were incubated with blocking buffer (5% skim milk + 100 ml TBST) at 37°C. Afterward, membranes were incubated with anti-TAZ antibody (Proteintech, China) overnight at 4°C, and incubated with HRP-linked secondary antibodies (Bioworld, U.S.A.) for 1 h at 37°C. The chemiluminescence method was used to detect the antibodies in the experiment (Bio-Rad, U.S.A.). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal reference.

The immunohistochemistry (IHC) procedures were performed using classical biotin–streptavidin–peroxidase staining protocols (22 (link)). TAZ expression was detected using anti-TAZ antibody (diluted 1:50; Catalog no: 23306-1-AP; Proteintech Group, Inc, United States). Five random high-power fields were chosen and more than 500 cells were analyzed per field for assessing TAZ expression. The sample TAZ expression scores were based on both the intensity of staining and the proportion of positively stained tumor tissues, with staining intensity scores being either 0 (negative), 1 (weak), 2 (moderate), or 3 (strong); and staining area scores being either 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%), or 4 (76–100%). The final staining score of each specimen was calculated as the product of staining intensity and the staining area scores. The final staining score of 2 or above was considered as positive TAZ expression whereas a staining score of less than 2 was considered as negative TAZ expression (23 (link)). The data were subjected to statistical analyses and the associated results are shown in the Supplementary Table 1.