Prrx 2

Total protein was extracted using the Whole Cell Lysis Assay kit (KeyGEN, Nanjing, China) and quantified using the BCA Protein Quantitation Assay kit (KeyGEN). Protein samples were subjected to sodium dodecyl sulfate gel electrophoresis and transferred to a polyvinylidene fluoride membranes. Subsequently, the membranes were incubated with 5% skimmed milk for 1 h, and probed with primary antibodies against MyoD (1:1000, Abcam, Cambridge, UK), MyHC (1:1000, Abcam), MyoG (1:1000, Abcam), Prrx-2 (1:1000, Abcam), CD63 (1:1000, Abcam), CD9 (1:1000, Cell Signaling Technology, Trask Lane Danvers, MA, USA), TSG101 (1:1000, Abcam), HSP70 (1:1000, Abcam), Calnexin (1:1000, Abcam), yes-associated protein 1 (YAP1, 1:1000, Abcam), OCN (1:2000, Abcam), OPN (1:1000, Abcam), RUNX2 (1:1000, Abcam), BMP2 (1:1000, Abcam) and GAPDH (1:1000, Abcam) at 4 °C overnight. After incubation with the appropriate secondary antibody, the protein bands were visualized using the ECL Detection Kit (KeyGEN).

IHC was performed using an immunohistochemical labeling kit (MaxVision Biotechnology, Fuzhou, Fujian, China) as previously described [10 (link)]. Briefly, paraffin-embedded sections of tumor specimens were labeled with primary antibodies against PRRX2 (1:100; Abcam), GCH1 (1:100; Abcam), and Ki-67 (1:100; Abcam). The immunohistochemical results were evaluated according to the German immunohistochemical score (GIS) [25 (link)].