Protein Expression Analysis by Western Blot

Cells were lysed in RIPA buffer (Beyotime, Jiangsu, China) containing a protease inhibitor cocktail, and the cell extracts were subjected to western blotting as previously described [19 (link)]. Primary antibodies were against PITX1 (1:500, Abcam, USA), cleaved-caspase 3, caspase 3, PARP, cleaved-PARP, FAK, p-FAK, Src, p-Src, Akt, p-Akt, PI3K, p-PI3K, E-cadherin, vimentin, β-catenin, p- β-catenin, Myc, TGS101, CD63, CD81, CD163, Flag, STAT3, ubiquitin, GAPDH and β-actin (1:1000, Cell Signaling Technology, USA), as well as CCL22 (1:1000, Abnova, Guangzhou, China). The secondary antibodies used were anti-Rabbit (CST, #7074, 1:1000) and anti-Mouse (Cell Signaling Technology, #7076, 1:1000). The protein band intensities were quantified using the Bio-Rad Molecular Imager ChemiDoc TM XRS + system.