Expression and bioactivity of wild-type LHCGR and LHCGR variants in vitro. (A) Western blot analysis of the protein expression of wild-type LHCGR and LHCGR variants in HEK-293T cells transfected with wild-type LHCGR (WT) and p.Ile585Leufs*16-, p.Arg283*-, and p.Asn377Asp-expressing vectors. (B) N-glycosidase F treatment of LHCGR and LHCGR variants. The indicated samples were treated with N-glycosidase F and detected by Western blotting. (C) The subcellular location of the wild-type LHCGR and LHCGR variants with and without hCG (1 × 105 ng/mL) stimulation. Magnification ×1200. White scale bar 10µm. (D) Response of cAMP to stimulation with increasing hCG concentrations in HEK-293T cells transfected with WT, p.Ile585Leufs*16, p.Arg283*, p.Asn377Asp, or empty vector. ★P < 0.05. (E) The ERK phosphorylation state after hCG stimulation (1 × 105 ng/mL) in HEK-293T cells transfected with WT, p.Ile585Leufs*16, p.Arg283*, p.Asn377Asp, or empty vector.
