Zeb1 3396

Cells were lysed and proteins extracted with RIPA buffer [10 (link), 54 (link)]. Protein concentration was measured using the Bradford method (Bio-Rad Laboratories). Samples were separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad Laboratories). The following antibodies were obtained from Santa Cruz Biotechnology Inc.: ADAR1 (SC-73408), p-AKT ser473 (sc-7985-R), AKT (sc-8312), pERK Tyr204 (sc-7383), and HRAS (C-20) (sc-520). ERK1/2 (#9102) and ZEB1 (#3396) antibodies were purchased from Cell Signaling Technology Inc., the PCNA (ab92552) antibody was from Abcam and the GAPDH (MAB374) antibody was from EMD Millipore Corp.

Total proteins were isolated by RIPA (Thermo Scientific, USA) from tissues and cell lines and subjected to western blotting analysis. Thirty micrograms of each lysate was electrophoresed in 10% SDS-PAGE gels, then transferred to PVDF membrane (Millipore, USA) and detected by using ECL kit (Beyotime Biotechnology, China). The anti-PTTG1 (ab79546) antibody and anti-p53 (ab28) antibody were procured from Abcam (Cambridge, UK). p21 (#2947), E-cadherin (#4065), Zeb1 (#3396), Vimentin (#3932), Snail (#3879), Slug (#9585), p-AKT (#9275), Akt (#9272) and GAPDH (D16H11) antibodies were all obtained from Cell Signaling Technology Inc. (Vebery, MA, USA). Horseradish peroxidase-conjugated anti-rabbit IgG, acting as the secondary antibody, was provided by Wuhan Boster Bio-engineering Co. Ltd., Wuhan, China. HE staining was employed to detect the lung metastases in rat models. Immunostaining was performed on BC tissue sections that had been previously confirmed for the pathological pattern by pathologists. The avidin-biotin-peroxidase method was performed to detect the expression and location of the target gene. The anti-PTTG1 primary antibody was used at a dilution of 1:100. An Olympus microscope was used to obtain images.