enance stage with establishment of cell–cell adhesion and a stationary phase. We investigated here whether Necl-4 also regulated the contact inhibition of cell movement and proliferation, because it binds protein-tyrosine phosphatase, non-receptor type 13, a putative tumor suppressor, through the cytoplasmic region of Necl-4, and inhibits the heregulin-induced activation of the ErbB2/ErbB3 signaling through PTPN13 and the phorbol ester-induced disassembly of hemidesmosomes. Necl-4 acts as a tumor suppressor and its expression is lost or markedly reduced in various human cancer cell lines. To investigate the role of Necl-4 in contact inhibition, we used cultured endothelial cells as a model cell line for several reasons. First, ECs have frequently been used 2 / 20 Regulation of Contact Inhibition by Necl-4 to study this phenomenon: under sparse conditions, ECs are highly sensitive to stimulation by growth factors, including VEGF, and their cellular status is comparable to moving and proliferating cells. When they reach confluence and establish firm cell–cell junctions, ECs lose the ability to respond to growth factors and switch to a quiescent condition. Second, ECs can be easily analyzed for tubulogenesis, a form of organogenesis, in vitro. Lastly, we have a substantial amount of information on nectins and Necls in ECs. We show here that Necl-4 serves as a novel regulator for contact inhibition of cell movement and proliferation at the initiation stage. Materials and Methods Antibodies, plasmids, and reagents Rabbit anti-Necl-1 polyclonal antibody, rat anti-Necl-2 mAb, and chicken antisera against Necl-5 mAb were prepared as described previously. Rabbit anti-Necl-3 pAb was raised against the 2nd loop of mouse Necl-3. Rabbit anti-Necl-4 pAb was raised against the cytoplasmic tail of mouse Necl-4. Alexa 488-conjugated isolectin B4, goat anti-vascular endothelial cadherin pAb, mouse anti-Necl-4/SynCAM4 mAb, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19768759 mouse anti-human Necl-5/CD155 mAb, rabbit anti-nectin-2 mAb, goat anti-nectin-3 pAb, mouse anti-vinculin mAb, Alexa 488-conjugated phalloidin, mouse anti-afadin/AF6 mAb, rabbit anti-Rap1 pAb, mouse anti-FLAG mAb, rabbit anti-FLAG pAb, rabbit anti-VEGF receptor 1 pAb, rabbit antiphospho-VEGFR2 pAb, rabbit anti-VEGFR2 pAb, rabbit anti-p44/42 MAPK pAb, rabbit anti-phospho-p44/42 MAPK pAb, rabbit anti-phospho-myosin phosphatase target subunit 1 /myosin-binding subunit pAb, rabbit anti-MYPT1/MBS pAb, mouse anti-Rac1 mAb, rabbit anti-PTPN13 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19769421 pAb, and mouse anti-actin mAb were purchased from the indicated suppliers. Fluorophore conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories and Merck Millipore. HRP-conjugated secondary antibodies were purchased from GE Healthcare Bioscience. 40,MedChemExpress 169939-93-9 6-Diamidino-2-phenylindole dihydrochloride was purchased from Nacalai Tesque, Inc.. pCAGIPuro-FLAG-Necl-4, pFLAG-CMV1-Necl-4-CP, and pFLAG-CMV1-Necl-4-EC were prepared as described. pCI-neo-VEGFR1 and pCI-neo-VEGFR2 were prepared as described. Human recombinant VEGF was purchased from Wako Pure Chemical Industries, Ltd.. Growth factor-reduced Matrigel matrix without phenol red was purchased from BD Biosciences. Y-27632 and fasudil were purchased from Merck Millipore. Cell culture and transfection experiment Primary cultures of human umbilical vein ECs were obtained from Lonza and maintained at 37C using Endothelial Cell Growth Medium 2 as described previously. Cells between passages 3 and 8 3 / 20 Regulation of Contact Inhibitio