Anti fsp1

The cells were lysed on ice using RIPA buffer containing protease inhibitors. The cytoplasmic extract (CE) buffer was composed of 1% Nonidet P-40, 150 mM NaCl, 50 mM Tris pH 8.0, 1 mM sodium orthovanadate and 5 mM NaF. The nuclear and cytoplasmic proteins were extracted according to the manufacturer's protocol, and proteins (30 µg/lane) were separated by 10% SDS-PAGE, subsequently electro-transferred onto PVDF membranes. The detection of blotted proteins was performed with anti-E-cadherin (1:500; cat. no. sc-8426), anti-type I collagen (1:1,000; cat. no. sc-59772), anti-Gli1 (1:1,000; cat. no. sc-20687), anti-Snail1 (1:1,000; cat. no. sc-271977), anti-GAPDH (1:1,000; cat. no. sc-47724) (Santa Cruz Biotechnology, Inc.) and anti-FSP1 (1:1,000; cat. no. ab124805; Abcam) antibodies. Primary antibodies were added to the membrane in 5% nonfat dry milk at 4°C overnight and were then incubated with a horseradish peroxidase-linked anti-rabbit or anti-mouse secondary antibody (1:2,000; cat. no. sc-2004, sc-2005; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Immunodetection was performed by chemiluminescence (ECL, Millipore, Billerica, MA, USA). GAPDH was used as an internal control. The gray levels of the blots were measured using ImageJ software version 1.48 (National Institutes of Health, Bethesda, MD, USA). The experiment was repeated three times.

Immunofluorescence staining was performed as described previously (15 (link)). HDM and TGF-β1 were added to the wells. The cells were incubated with anti-E-cadherin (1:50; cat. no. sc-8426), anti-Gli1 (dilution 1:100; cat. no. sc-20687; Santa Cruz Biotechnology, Inc.), and anti-FSP1 (1:150; cat. no. ab124805; Abcam) antibodies at 4°C overnight, and were subsequently stained with secondary antibodies conjugated with Alexa Green 488 (1:1,000; cat. no. A-11001; Molecular Probes) or with Cy3-labelled secondary antibodies (1:1,000; cat. no. 111-136-144; Jackson ImmunoResearch Laboratories) for 1 h at room temperature. Cells were also stained with DAPI. The slides were visualized with a Zeiss Axio Imager 2 microscope (Carl Zeiss AG).