Anti tgfb1 antibody

Samples were fixed in 4% buffered formalin (Sigma-Aldrich) for 10 min at room temperature. The samples were then incubated with 0.1% Triton-X100 (USB corporation, Cleveland, OH, USA) for 5 min and subsequently immersed in 2% horse serum (Hyclone, South Logan, UT, USA) for 30 min at room temperature. The samples were stained with anti-Tgfb1 antibody (Abcam, Cambridge, UK) overnight at 4 °C. A biotinylated goat-anti rabbit antibody (Santa Cruz Biotechnology, Dallas, TX, USA) was used as a secondary antibody. Fluorescence labeling was performed with Streptavidin, Rhodamine Red-X conjugate (Invitrogen, MD, USA). Nuclei were stained with 4’, 6-diamidino-2-phenylindole (DAPI: Tocris Bioscience, Bristol, UK). The samples were then observed under an Apotome.2 (Carl Zeiss, Jena, Germany) fluorescence microscope.

The protein levels of DNMT1, TGFb1, USF1 and SREBP1 were analyzed by immunoblotting using anti-DNMT1 antibody (Abcam, no. ab188453), anti-TGFb1 antibody (Abcam, no. ab92486), anti-USF1 antibody (Santa Cruz Biotechnology, no. sc-229) and anti-SREBP1 antibody ((R&D Systems, no. NB100-60545SS)). Expression levels of beta-actin were used for normalization. The protein level of beta-actin was analyzed using anti-beta-actin antibody (Abcam, no. ab8227). Wide-view prestained protein size marker III was used as size marker of DNMT1 protein (Wako, no. 230-02461).