Secretion of LHCGR recombinant proteins. Cell culture supernatants (M) at a), 24h and b), 48h following mock transfection or transfection with LHCGR cDNAs were centrifuged and concentrated, and corresponding whole cell extract (E), hypotonic (HE) and detergent (DE) extract proteins were resolved via SDS-PAGE before detection by anti-FLAG antibody in Western blots. Tissue culture supernatants (M) prepared from c), 48h, 144h and 168h following transfection; the results shown in d), demonstrate abrupt increase in secretion of the recombinant LHCGR on D4 (96h) following transfection; e) and f), quantitative analysis of secreted proteins through seven days post-transfection showing the relative amounts of each secreted recombinant protein compared to cytosolic plus membrane associated recombinant protein. The cell-associated and the supernatant LHCGR values were derived by comparing the expression of each recombinant protein from the same culture volume (Figure 3f). LHCGR number 1, 2 and 3 represent recombinants R1, R2 and R3 as shown in Figure 1. The concentration of the LHCGR recombinants in the supernatant was highest on day 4. The mean expression of R1, S (P < 0.001, R2, S (P < 0.001) and R3, S (P < 0.0005) was significantly higher (indicated by ***) compared to that of corresponding cell-associated LHCGR on D4 and D5.