Antigen retrieval was performed by boiling slides in EDTA buffer (pH 9) for 20 moments, and the slides were cooled on bench top for 20 moments. receptor tyrosine kinase family has four users: EGFR (ErbB1), ErbB2, ErbB3, and ErbB4 . You will find seven ligands for EGFR: epidermal growth factor (EGF), LY278584 transforming growth factor- (TGF-), heparin-binding EGF-like growth factor (HB-EGF), betacellulin (BTC), amphiregulin (AREG), epiregulin (EREG), and epigen (EPGN) , . You will find two ligands for ErbB3: heregulin1 (HRG1) and heregulin2 (HRG2), which are the type I and II isoforms of neuregulin family (NRG1-4) . The seven EGFR ligands demonstrate different binding affinities to EGFR and can be divided into two groups: EGF, TGF-a, BTC, and HB-EGF with high affinity and the others with low affinity , . Their capacities to induce EGFR dimerization are also different . Consequently, they induce different biological effects even in the same cell collection . Although four of the EGFR ligands have a higher affinity than the other three, the expression levels of the high-affinity ligands are not as high as those of the low-affinity ligands in certain malignancy cells , . As a result, the specific ligand that eventually occupies EGFR on malignancy cells is not obvious. In addition, EGFR can form a homodimer or a heterodimer with ErbB3 , creating further ligand binding complexity. According to the rotation model of EGFR-ErbB3, EGFR and ErbB3 form a heterodimer before the ligands bind , , indicating that both EGFR ligands and ErbB3 ligands could bind to the EGFR-ErbB3 heterodimer simultaneously. The effect on cells by different combinations of EGFR and ErbB3 ligands binding to EGFR-ErbB3 heterodimer is not understood . It is well known that EGFR mutation (EGFRmut) plays an important role in cancer development , , . In nonCsmall cell lung malignancy (NSCLC) cells, the deletion of five amino acids (E746-A750del) and point mutation (L858R) of EGFR are associated with the development and maintenance of this disease , , , . Although mutations of EGFR increase their kinase activity, the mutants still need ligand stimulation for further activation , . Currently, it is not obvious which ligand is responsible for the initiation and progression of NSCLC with EGFRmut. It is also not clear whether the EGFRmut-EGFRmut homodimer or EGFRmut-ErbB3 heterodimer is the driver for NSCLC development. In this study, we investigated which EGFR ligand or ErbB3 ligand is responsible for LY278584 NSCLC proliferation. We also investigated the mechanism behind their action. Materials and Methods Cell Lines and Materials All cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and the cell lender of the Chinese Academy of Sciences (Shanghai, China). The cells were expanded when they showed PRKACG up. Cells were aliquoted into 20 to 30 vials and kept in liquid nitrogen after being found mycoplasma-free using two test packages (Mycoalert Mycoplasma Detection Kit LT07-218 from Lonza and PCR Mycoplasma Test Kit K0103 from HuaAn). The Cell Counting Kit-8 (CCK8) was purchased from Dojindo (Tokyo, Japan). The antibodies of antiCphospho-AKT (cat. no. 4060), antiCphospho-ERK1/2 (cat. no. 9101), anti-ERK (cat. no. 9102), anti-HER3/ErbB3 (cat. no. 12708), anti-rabbit IgG (H + L), F(ab)2 Fragment (Alexa Fluor 488) (cat. no. 4412), protein-A agarose beads (cat. no. 9863), and the rabbit polyclonal anti-EGFR LY278584 antibody (cat. no. 2232) were purchased from Cell Signaling Technology (Danvers, MA). The antibodies of anti-EGFR (cat. no. ab52894), anti-ErBb3 (cat. no. ab20161; cat. no. ab93739), anti-Mouse IgG H&L (Alexa Fluor 647) (cat. no. ab150115), and anti-EGF (cat. no. ab9695) were purchased from Abcam (Cambridge, MA). The antibodies of anti-Betacellulin (cat. no. bs-12864R) and anti-Epigen (cat. no. bs-5767R) were purchased from Bioss (Beijing, China). The antibodies.