hCG signals hESC proliferation via LHCGR. (a) Pluripotent H9 hESC, EBs and NPC lysates were analyzed by immunoblot with (i) an affinity-purified anti-human LH/hCG receptor polyclonal antibody against the N-terminal 15-38 amino acids; (ii) a monoclonal antibody against Oct-3/4 (C-10; against 1-134 amino acids of human Oct-4), and (iii) monoclonal antibodies against human β-actin and GAPDH [11, 18]. Molecular-weight markers are shown on the left of immunoblots. (b) Protein from cell lysates of hESCs were analyzed with immunoblot by using a polyclonal antibody against LHβ . (c) hESCs were treated with lipofectamine (control), lipofectamine + LHCGR sense oligonucleotides, or lipofectamine + LHCGR antisense-P oligonucleotides for 6 days, and the cells were then counted by using the trypan blue method. Results are expressed as mean ± SEM, n = 4; significant differences between groups is indicated by different letters, P < 0.05. (d) Schematic of the LHCGR activation site and binding site of rabbit polyclonal antibody against amino acids 15 to 38 of the extracellular binding domain of LHCGR. (e) hESCs grown in six-well plates coated with Matrigel in mTeSR1 media were treated for 6 days with (i) hCG (500 mIU/ml; lane A); (ii) increasing concentrations of the affinity-purified rabbit polyclonal antibody against amino acids 15 to 38 of the extracellular binding domain of LH/hCG receptor (1:1,000, 1:200, 1:100; lanes B, C, and D, respectively); and (iii) in combination with (500 mIU/ml of hCG (lanes E, F, and G). Cell number was counted by using the trypan blue assay. Results are expressed as mean ± SEM, n = 3; *P < 0.05; **P < 0.005 compared with day 6 control. (f) hESCs were cultured in growth factor-free TESR1 medium ± hCG (500 mIU/ml) for 6 days, and cell number was measured by using the trypan blue assay. Results are expressed as mean ± SEM, n = 3; **P < 0.005 compared with 6 day control.