Protein Expression Analysis of Cancer Signaling

Whole cell lysates were prepared using RIPA buffer supplemented with protease and phosphatase inhibitor cocktail (Thermo, USA) and culture supernatants were concentrated using Microcon centrifugal filters (Millipore, USA). Western blot was performed as previously described [17 (link)]. Primary antibodies against the following proteins were used: CTHRC1 (ab192778, Abcam, USA), p-c-Raf (9427, CST, USA), p-MEK1/2 (9154, CST, USA), p-ERK1/2 (4370, CST, USA), ERK1/2 (4695, CST, USA), p-FRA-1 (5841, CST, USA), FRA-1 (5281, CST, USA), cyclinD1 (2978, CST, USA), snail1 (3879, CST, USA), and MMP14 (13130, CST, USA). α − Tubulin (T9026, Sigma-Aldrich, USA) was used as a loading control.