Non flat milk

As described previously (Lin et al. 2018) . Proteins were extracted from cultured stromal cells with lysis buffer. The BCA reagent kit (Applygen, Beijing, China) was used to measure the concentration of proteins. The protein samples were separated by 12% SDS-PAGE gel and transferred onto PVDF membranes (Millipore). PVDF membranes were blocked with 5% non-flat milk (Sangon, Shanghai, China) and followed by probing with the corresponding antibodies for CYPA (final concentration: 1 µg/mL; Abcam), p-Stat3 (final concentration: 12 ng/mL; Cell signaling), T-Stat3 (final concentration: 24 ng/mL; Cell signaling), Vimentin (final concentration: 0.27 µg/mL; Proteintech), Cytokeratin 18 (CK18, final concentration: 0.3 µg/mL; Proteintech), β-Tubulin (final concentration: 0.1 µg/mL; Proteintech) and Gapdh (final concentration: 0.2 µg/mL; Santa Cruz) overnight at 4°C. After washing, the matched secondary antibodies conjugated with HRP (final concentration: 0.1 µg/mL; Elabscience) were incubated PVDF membranes. Signals were developed with the ECL kit (Sangon, Shanghai, China).

Western blot was performed as described previously (Liang et al. 2014) (link). Briefly, proteins for Western blot were extracted from cultured stromal cells by homogenization lysis buffer including 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100 and 0.25% sodium deoxycholate. The concentration of proteins was measured by BCA kit (Applygen, Beijing, China). The samples (10 μg per lane) were separated by 10% SDS-PAGE gel and transferred onto PVDF membranes (Millipore) followed by blocking with 5% non-flat milk (Sangon, Shanghai, China). PVDF membranes were probed with the corresponding antibodies for FADS3 (ABclonal, Wuhan, China) and β-Actin (Cell Signaling) overnight at 4°C. Membranes were then incubated with the matched secondary antibodies conjugated with HRP (1:5000). Signals were detected with the ECL kit (Pierce Biotechnology).