Sab2108306

Following drying in a 60°C oven overnight, the tissue sections were deparaffinized in xylene and rehydrated in a gradient ethanol series. For antigen retrieval, the sections were microwaved in 10 mM citrate buffer (pH 6.0) for 20 min. The sections were then incubated in 5% bovine serum for 1 h at 37°C. A combination of two primary antibodies, rabbit polyclonal anti-SOX4 (SAB2108306; 1:100 dilution; Sigma-Aldrich Merck KGaA, St. Louis, MO, USA) and mouse monoclonal anti-p180 (ab24751; 1:50 dilution; Abcam, Cambridge, UK), was used for incubating the sections overnight at 4°C, while for negative controls PBS was used in the place of the antibody. The sections were then incubated with a mixture of two secondary antibodies, Alexa Fluor-594 donkey anti-rabbit IgG (ab150076; 1:500 dilution; Abcam) and Alexa Fluor-488 donkey anti-mouse IgG (ab150105; 1:500 dilution; Abcam) for 4 h at room temperature, followed by DAPI incubation for nuclear staining. Double immuofluoresence images were acquired using a confocal laser-scanning microscope (C1; Nikon, Tokyo, Japan).

Western blotting was performed using standard protocols. In brief, total protein was extracted from frozen lung tissue samples and mixed with loading buffer. Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDA-PAGE) (100 V for 3 h) and transferred to polyacryl-amide difluoride (PVDF) membranes (100 V for 50 min). Membranes were then blocked in 5% skimmed milk for 2 h at room temperature. Membranes incubated with rabbit anti-SOX4 (SAB2108306; 1:800 dilution; Sigma-Aldrich Merck KGaA) or mouse anti-EZH2 (612666; 1:1,000 dilution; BD Transduction Laboratories, San Jose, CA, USA) were shaken overnight at 4°C. The next day, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibody (1:5,000 dilution; Proteintech, Rosemont, IL, USA) for 2 h after washing three times in Tris-buffered saline and Polysorbate 20, and developed using enhanced chemiluminescence reagents (Thermo Scientific Pierce; Thermo Fisher Scientific, Waltham, MA, USA). Densitometry values were detected for all bands and standardized relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for each sample.