Time course analysis of the expression of the LHCGR recombinants. Three recombinant LHCGR proteins expressed in a), the cytosolic fractions (hypotonic lysis) and b), those associated with the cell membrane (sequential hypotonic and detergent lysis). Equal amounts of protein for each 24h time-point (D1 to D7) and each recombinant (R1, R2 and R3) were resolved by 8% SDS-PAGE and, following transfer of the proteins via Western blotting, were first probed with anti-FLAG monoclonal antibody and subsequently with a β-actin specific monoclonal antibody. The estimated molecular mass (Mr) of recombinants 1, 2 and 3 were 45K, 37K and 32K, respectively. LHCGR number 1, 2 and 3 represent recombinants R1, R2 and R3 as shown in Figure 1. The density of each band was measured, normalized to the corresponding β-actin value and the expression of each LHCGR recombinant in c), cytosolic (hypotonic) and d), membrane-bound (detergent) fractions were plotted to evaluate the quantitative expression.