Immunohistochemistry (IHC) was performed in Chinese lung and breast cancer tissues using the furin antibody 31 , 32 (link), 34 (link). Briefly, the de-paraffinized and re-hydrated tissue sections were treated in the 10 μM sodium citrate buffer at 95 °C for 12 minutes for antigen retrieval. The treated tissue slides were further incubated in the solution with 3% H2O2 for quenching the endogenous peroxidase. After blocking with 5% bovine serum albumin (BSA), the tissue slides were incubated with the primary antibody by overnight at 4 °C (rabbit monoclonal antibodies, 1:200; cat #: ab183495, Cambridge, CB2 0AX, UK) and then the biotin-conjugated secondary antibody (cat #: SP-9000, ZSGB-Bio, CN) for 60 minutes at room temperature. The bound secondary antibody was visualized by sequentially incubating with the Streptavidin-conjugated horseradish peroxidase (HRP) and its substrate diaminobenzidine (ZLI-9017, ZSGB-Bio, CN).
Cordycepin (CD) was purchased from Chengdu Must Bio-Technology Co.Ltd (Chengdu, Sichuan, P. R. China). Western blotting analysis for furin expression was performed in lung cancer cell line H1975 and breast cancer cell line BT549 with CD treatments (0, 10 µm, 20 µm, 40 µm) for 24 hours. The furin antibody for western blotting was used as same as IHC (rabbit monoclonal antibodies, cat #: ab183495, Cambridge, CB2 0AX, UK). The semi-quantitative RT-PCR for furin expression was performed in breast cancer cell line BT549 with CD treatments (0, 10 µm, 20 µm, 40 µm) for 24 hours. RT-PCR primers for furin were as follows, 5'-tgtggtgtaggtgtggccta-3' (RT-furin-L), 5'-gctgatggacagcgtgtaga-3' (RT-furin-R). GAPDH was served as internal control. All experiments were repeated at three times.
Cordycepin (CD) was purchased from Chengdu Must Bio-Technology Co.Ltd (Chengdu, Sichuan, P. R. China). Western blotting analysis for furin expression was performed in lung cancer cell line H1975 and breast cancer cell line BT549 with CD treatments (0, 10 µm, 20 µm, 40 µm) for 24 hours. The furin antibody for western blotting was used as same as IHC (rabbit monoclonal antibodies, cat #: ab183495, Cambridge, CB2 0AX, UK). The semi-quantitative RT-PCR for furin expression was performed in breast cancer cell line BT549 with CD treatments (0, 10 µm, 20 µm, 40 µm) for 24 hours. RT-PCR primers for furin were as follows, 5'-tgtggtgtaggtgtggccta-3' (RT-furin-L), 5'-gctgatggacagcgtgtaga-3' (RT-furin-R). GAPDH was served as internal control. All experiments were repeated at three times.
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