Sulf1

Cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris–HCl pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with 1 × protease inhibitor cocktail (Complete Mini, Roche), 0.2 mM sodium orthovanadate, 20 mM sodium fluoride, and 1 mM phenylmethylsulfonyl fluoride (SIGMA); 25 μg of protein lysate was loaded onto 8% SDS PAGE gels. Immunoblots were performed with antibodies against ITG11A1 (Abnova, 1:700), COL11A1 (Abcam 1:1,000), MMP13 (Thermo Scientific, 1:500), INHBA (Abcam, 1:500), SULF1 (Abcam, 1:500), EPYC (SIGMA, 1:2,000), TNFSF4 (BioLegend, 1:300) and ERK2 (Santa Cruz Biotechnology, 1:2,000) overnight at 4°C.

Cells were harvested on ice by using NETN (150 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl, and 0.5% NP40) buffer supplemented with protease inhibitors, and lysates were incubated with antibodies for overnight at 4°C. Immunocomplexes were precipitated with protein A/ G (Roche Applied Science) for 3 hr at 4°C. The beads were washed four times with NETN buffer. Proteins were then eluted from the beads by boiling in sample buffer for 5 min and then analyzed by electrophoresis on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Whole-cell lysates were analyzed with the following antibodies: pcMET (#3077 Cell Signaling), cMET (#8198 Cell Signaling), CD44v6 (BBA13 R&D), pERK 1/2 (GTX59568 #9101 Cell Signaling), ERK 1/2 #4695 Cell Signaling, H3K27me3 ab6002 Abcam, Histone 3 (GTX122148, #9715 Cell Signaling), ß-Actin (sc-47778 Santa Cruz Biotechnology), EZH2 (#3147, #5246 Cell Signaling), pP38 (#9211 Cell Signaling), P38 (sc-7149 Santa Cruz Biotechnology), SULF1 (ab32763 Abcam), and 10E4 (#370255 S, amsbio). The image detection was analyzed by ChemiDoc Touch Imaging System (Bio-Rad) and Image Lab software (Bio-Rad).