We have reported that knockdown of Necl-4 decreases vascular endothelial growth factor (VEGF)induced phosphorylation of extracellular signal-regulated kinase (ERK) without affecting phosphorylation of VEGF receptor 2 (VEGFR2) in sparsely cultured human umbilical vein endothelial cells (HUVEC5). However, the underlying molecular mechanism is unknown. Compared with control HUVECs, VEGFinduced phosphorylation of phospholipase Cy (PLCy), c-Raf, mitogen-activated protein kinase/ERK kinase (MEK) and ERK were all inhibited in Necl-4-knockdown HUVECs. However, VEGF-induced internalization of VEGFR2 was not different between control and Necl-4-knockdown HUVEC5. We have reported that protein-tyrosine phosphatase, non-receptor type 13 (PTPN13) and Rho-associated kinase (ROCK) are involved in the Necl-4-knockdown-induced inhibition of the VEGF-induced activation of Racl. However, the effects of Necl-4-knockdown on VEGF-induced phosphorylation of VEGFR2 and ERK were not affected either by knockdown of PTPN13 or by ROCK inhibitors. These results suggest that Necl4 enhances VEGF-induced activation of PLCy c-Raf MEK ERK pathway without affecting the phosphorylation and internalization of VEGFR2. (C) 2017 Elsevier Inc. All rights reserved.