Figure 3. Enzymatic analyses of the recombinant human SCD2 expressed in COS-7 and Sf9 cells.

(A) Analysis of SCD activity from the recombinant human SCD1 and SCD2 enzymes transiently expressed in COS-7 cells. COS-7 cells transiently transfected with either the hSCD1 expression vector, the hSCD2 expression vector, or empty vector were lysed with cell-harvesting buffer containing multiple protease inhibitors (1×Complete™) after 48 h of transfection, and were processed for SCD enzyme assays. (B) Analysis of SCD activity from the recombinant human SCD1 and SCD2 enzymes expressed in Sf9 insect cells. Sf9 cells infected with the hSCD1 or the hSCD2 recombinant baculovirus, as well as wild-type baculovirus were harvested, after 72 h of infection, in the same harvesting buffer used for COS-7 cells, and processed for SCD activity assays. Desaturase activities were determined by conversion of radiolabelled palmitoyl-CoA (C_16:0) and stearoyl-CoA (C_18:0) into the corresponding mono-unsaturated fatty acids (indicated by an arrowhead). The SCD enzyme activities were quantified by PhosphorImaging from three independent experiments, and are expressed as pmol/min per mg of protein.