Mek1 2

Platelet-derived growth factor (PDGF) was purchased from Proteintech (Wuhan, China); U0126 was provided by Selleck Chemicals (Shanghai, China); fetal bovine serum (FBS), RPMI-1640, and penicillin-streptomycin solution were purchased from Gibco (Thermo Fisher Scientific, Waltham, MA); ovalbumin (OVA) was obtained from Sigma-Aldrich (Saint Louis, MO, United States); and dexamethasone (DEX) was purchased from Xianju Pharmaceuticals (Zhejiang, China). p-ERK1/2, ERK1/2, and HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA); p-MEK1/2, MEK1/2, and β-actin antibodies were obtained from Affinity (Changzhou, China); PCNA, α-SMA, and GAPDH antibodies were purchased from Proteintech (Wuhan, China); and RIPA lysis buffer and BCA protein assay kit were obtained from Beyotime (Shanghai, China).

Antibodies against PKG I, EGFR, p-EGFR (T693), ERK1/2, VASP, c-Raf, MEK1/2, p-VASP (Ser 239), p-c-Raf (Ser338), p-Erk1/2 (Thr202/Tyr204), MEK1/2 (Ser217/Ser221), and Grb2 were purchased from Affinity Biosciences (Affinity Biosciences, OH, USA). Antibodies against p-EGFR (Tyr1068) was purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against Sos1 was purchased from Santa Cruz (Santa Cruz, CA, USA). Antibodies against p-Thr/ser was purchased from Abcam Biotechnology (Abcam, Cambridge, UK). Antibody against β-actin was purchased from Proteintech (Proteintech, Chicago, USA). EGF was purchased from PeproTech (Rocky Hill, NJ, USA), 8-Br-cGMP, and Rp-8-Br-cGMPS were purchased from Biolog Biotechnology (Bremen, Germany) (Table 1).

Radio-Immunoprecipitation Assay (i.e., RIPA) buffer (Beyotime Biotechnology, CA, China) supplemented with 1 nM of benzylsulfinyl fluoride was added to extract total proteins from the cells or tumor samples, followed by a bicinchoninic acid (i.e., BCA) assay (Beyotime) to quantify the protein concentrations. The proteins (30 μg) were separated using a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (i.e., SDS-PAGE) gel, then transferred to polyvinylidene fluoride membrane (EMD Millipore, Burlington, MA, USA). The membrane was blocked for 2 h with non-fat milk followed by primary antibody incubation, including E-cadherin (1:1000; Abcam; No. ab231303), N-cadherin (1:1000; Abcam; No. ab18203), ERK1/2 (1:1000; Abcam; No. ab184699), p-ERK1/2 (1:1000; Abcam; No. ab223500), MEK1/2 (1:500; Affinity; No. AF6385), p-MEK1/2 (1:500; Affinity; No. AF8035), and GAPDH (1:1000; Proteintech; #60,004–1-1G) at 4 °C overnight. The blots were washed with TBS-T and treated with horseradish peroxidase-conjugated polyclonal goat anti-rabbit immunoglobulin G (IgG; 1:1000; No. A0208) or polyclonal goat anti-mouse IgG (1:1000; Beyotime; No. A0216) for 1 h at ambient temperatures. An enhanced chemiluminescence kit (Beyotime; WBKLS0100) was used to assess the protein bands.