NHERF-2 regulates cell cycle in ECs. HUVECs transfected with control-scrambled or NHERF-2 siRNA were synchronized and treated with or without VEGF 10 ng/mL for 20 hours and then subjected to (A) PI staining for cell-cycle determination, percentage of S+G₂/M phase population was calculated from 30 000 cells of each group (n = 3); or (B) nuclear lysates were collected and subjected to Western blot analysis with the use of c-Myc Ab and lamin B as control (n = 3); or (C) whole-cell lysates collected and subjected to Western blot analysis with Abs against pRb, Cyclin A, Cyclin B1, Cyclin D1, p21, p27 (N = 3). (D) HUVECs transfected with control-scrambled or NHERF-2 siRNA were synchronized and treated with or without VEGF 10 ng/mL for 12 hours and then cross-linked chromatin-protein complexes were isolated with βCat Ab as well as control immunoglobulin (Neg). After reverse cross-linking, DNA fragments were isolated, and the cyclin D1 promoter was amplified by PCR with the use of promoter-specific primers and run on a 2% agarose gel. The Input sample represents 5% of total chromatin DNA. Representative gel is shown with band intensities normalized with respect to control siRNA-transfected cells without VEGF (n = 3). When applicable, statistical significance was determined with 2-sided Student t test, and a value of P < .05 was considered significant.