Protein expression was assessed in samples from both HCC cell lines and resected patient tissues by Western immunoblotting as described previously 38 (link). The primary antibodies used were: TLX3 (ab184011; Abcam), p-STAT3 (9145; Cell Signaling Technology), STAT3 (9139; Cell Signaling Technology), SNAI1(3895; Cell Signaling Technology), E-cadherin (14472; Cell Signaling Technology), N-cadherin (4061; Cell Signaling Technology), Vimentin (5741; Cell Signaling Technology) and β-actin (8457; Cell Signaling Technology). All protein expression was normalized toβ-actin after densitometric scanning.
Co-immunoprecipitation (CO-IP) was conducted to determine whether TLX3 protein was bound with STAT3 protein according to the protocol reported previously 37 (link). Briefly, HCC cells were lysed with immunoprecipitation buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP40, 1 mM ethylenediaminetetraacetic acid, 10 mM sodium butyrate) containing protease inhibitors. The cell lysate was incubated with the antibody against TLX3 (ab184011; Abcam) overnight at 4˚C and protein A/G-agarose beads were added. The mixture was shaken overnight at 4˚C and then rinsed with immunoprecipitation buffer. The supernatant was analyzed by Western immunoblotting with the STAT3 antibody (9139; Cell Signaling Technology).
Co-immunoprecipitation (CO-IP) was conducted to determine whether TLX3 protein was bound with STAT3 protein according to the protocol reported previously 37 (link). Briefly, HCC cells were lysed with immunoprecipitation buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP40, 1 mM ethylenediaminetetraacetic acid, 10 mM sodium butyrate) containing protease inhibitors. The cell lysate was incubated with the antibody against TLX3 (ab184011; Abcam) overnight at 4˚C and protein A/G-agarose beads were added. The mixture was shaken overnight at 4˚C and then rinsed with immunoprecipitation buffer. The supernatant was analyzed by Western immunoblotting with the STAT3 antibody (9139; Cell Signaling Technology).
