Anti vimentin ab92547

Western blotting was performed as described previously (29 (link)). Briefly, cells were harvested and lysed in RIPA buffer supplemented with phosphatase (sodium fluoride, sodium molybdite, sodium pyrophosphate) and complete Mini, EDTA-free protease inhibitors (Roche). 15–25 μg total protein (depending on different proteins) were separated by SDS-PAGE and then transferred to polyvinylidene difluoride membranes (Roche). For each gel, the same amount of protein was loaded per lane. Membranes were then blocked in 5% non-fat milk in Tris-buffered saline-Tween for1 h. The immunoblotting was performed by incubation at 4°C overnight with the following primary antibodies: anti-PD-L1 antibody (13684) (Cell Signaling Technology), anti-E-cadherin (ab15148) (Abcam), anti-vimentin (ab92547) (Abcam) and β-actin (Cell Signaling Technology), which was used as an endogenous protein for normalisation. Blots were then washed and incubated with a 1:1000 dilution of anti-Rabbit IgG H&L (HRP)-conjugated secondary (Cell Signaling Technology) or a 1:1000 dilution of anti-mouse IgG H&L (HRP)-conjugated secondary (Cell Signaling Technology). Signals were detected by enhanced chemiluminescence Plus reagents (PerkinElmer) and images were captured using a Licor Odyssey System. Signal quantification was obtained using Image Studio Lite software (Version 5.2.5). All experiments were performed in triplicate.

The human PTC cell lines K1 and TPC-1 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). RPMI-1640, Dulbecco's modified Eagle's medium and fetal bovine serum (FBS) were purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Anti-FKBP51 (ab46002) used in immunohistochemistry (IHC), anti-IκBα (ab32518), anti-N-cadherin (ab76011), anti-Vimentin (ab92547), anti-β-catenin (ab32572) and anti-MMP9 (ab76003) were purchased from Abcam (Cambridge, UK). Anti-p65 (sc-8008), anti-FKBP51 (sc-13983), anti-GAPDH (sc-47724), anti-α-tubulin (sc-53646), secondary antibody anti-mouse IgG-horseradish peroxidase (HRP; sc-516102) and anti-rabbit IgG-HRP (sc-2372) used in western blotting were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Anti-TGF-β1 (3709) and anti-histone 3 (12164) was purchased from Cell Signaling Technology (Danvers, MA, USA). Ammonium pyrrolidine dithiocarbamate (PDTC; T3147) was purchased from Target Molecule Corp. (Wellesley Hills, MA, USA).