NANOG expression in HUVECs and tumor cell lines. (A) HUVECs were starved of growth factors and serum for 14 hours, then left untreated or treated with increasing concentrations of WNT3A (10, 20, or 30 ng/mL) for 6 hours. Total protein lysates prepared from tumor cell lines were subjected to WB analysis with the indicated antibodies. (B) Unstarved HUVECs were left untreated or treated with LiCl (20 ng/mL) or WNT3A (50 ng/mL) for 6 hours. Total cell lysates were analyzed by WB analysis with the indicated antibodies. Anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to determine equal protein loading. (C) Total RNA prepared from HUVECs left untreated or treated with WNT3A was subjected to RT-PCR for NANOG, FLK1, and GAPDH. Each experiment was repeated ≥ 3 times.