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. 2021 Nov 25;22(23):12735. doi: 10.3390/ijms222312735

Figure 3.

Figure 3

Production and heterodimerization in mutant and wild-type luteinizing hormone (LH) β and bioactivity of mutant and wild-type LH. Panel (A) shows low intracellular and secreted levels of mutant LH beta subunit in transfected cells. Western blot analysis was performed on cell lysate (lanes 1 through 3) and culture medium (lanes 4 to 6) of human embryonic kidney (HEK) 293T cells: cells producing the LHα subunit and the wild-type LH beta subunit (lanes 1 and 4), cells producing the LH alpha subunit and the mutant LHβ subunit (lanes 2 and 5), and mock-transfected cells (lanes 3 and 6). An anti–β-actin antibody was used as a loading control (lanes 1 through 3). Identical volumes of culture medium, concentrated by a factor of about 20 for wild-type LHβ (lane 4) and about 400 for mutant LHβ (lane 5), were loaded. Wild-type LHβ and mutant LHβ were immunodetected with an anti–hCGβ antibody (Abcam) displaying strong cross-reactivity. Panel (B) shows low levels of dimerization of the alpha subunit and mutant beta subunit of LH. Coimmunoprecipitation experiments were performed on cell lysates from COS-7 cells producing the α-V5 construct and either wild-type or mutant LHβ. Immunoprecipitation was performed with the use of anti-V5 antibody (lanes 4 and 5) or with a nonimmune immunoglobulin as a control (lane 3). This was followed by immunodetection of wild-type LHβ and mutant LHβ, with the use of a polyclonal anti–hCGβ antibody (Abcam), or of the α-V5 construct, with an anti-V5 antibody. An anti–β-actin antibody was used as a loading control (lanes 1 and 2). Panel (C) shows markedly lower levels of mutant LH bioactivity in HEK 293 cells expressing the human LH receptor. The secreted levels of wild-type LH were quantified by means of immunofluorometric assay and used to generate a dose–response curve. Comparative quantification of secretion of wild-type and mutant LHβ was carried out through Western blot analysis (Panel (A), reflecting one representative experiment). HEK 293 cells expressing the human LH receptor were stimulated with concentrated culture medium containing a similar amount of either wild-type LH or mutant LH beta. Concentrated culture medium from mock-transfected cells were used as a negative control. The mean results are shown for three independent experiments using each of three doses of mutant or wild-type LH to stimulate cyclic AMP production. I bar indicates standard deviations. From [46], with permission.