Ab178886

Proteins were extracted from samples using RIPA Kit (Beyotime Biotechnology, China). The protein concentration was quantified using Bradford method. Proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and thereafter transferred to PVDF membrane which was blocked in Tris‐Buffered Saline Tween (TBST) containing 0.1%Tween‐20 and 5% low‐fat milk for 1 h. The primary antibody was added and incubated overnight at 4°C and then the secondary antibodies were added and incubated at room temperature for 1 h. Lastly, the ECL solution was applied to the membranes, and the membranes together with a film were placed in a visualizer for further analysis. The ImageJ software was used to measure the intensities. Primary antibodies for NRAS (ab154291), BRAF (ab151286), MEK1/2 (ab178886), pMEK1/2 (phospho s218, s222 and s226, ab78132), ERK1/2 (ab17942), pERK1/2 (phospho thr202 and thr204, ab214362), and GAPDH (ab9485) were all purchased from Abcam, whereas antibodies for PI3K‐p110α (#4249), PI3K‐p110β (#3011s), AKT (#9272), pAKT‐ser473 (#4060), pAKT‐thr308 (#2965), PTEN (#4005), Cyclin D1 (#2926), and p27 (#3686) were purchased from Cell Signaling. HRP labeled goat anti‐mouse and goat anti‐rabbit as secondary antibodies were purchased from Beyotime Biotechnology.

Secreted protein levels of human pro-collagen-I α1, interleukin-6 and monocyte chemoattractant protein 1 were determined by commercial ELISA kits (Abcam, Bristol, UK, ab210966, ab178013 and ab178886) according to the manufacturer’s instructions. Protein levels were normalized to the wet weight of explanted samples and expressed as pg/mg tissue.