Figure 5. Cross-linking analyses of human SCD1 and SCD2 expressed in COS-7 cells.

COS-7 cells were transiently transfected with each of the FLAG-tagged version of hSCD expression vector or empty vector, and were treated with 0, 0.5 and 1.0 mM of DSP respectively for 30 min at 37 °C, followed by Western blot analyses using anti-FLAG antibodies, or enzyme assay using [¹⁴C]stearoyl-CoA as a substrate. (A) Detection of different SCD moieties by Western blot analyses. The same blot was stripped and re-probed with anti-GAPDH antibodies to provide an internal control for protein loading. The position of different moieties of each enzyme is indicated by arrows, and degraded product is shown by arrowheads. A sample from mock-transfected COS-7 cells was used as a negative control (C). (B) Quantification of different moieties of hSCD1 and hSCD2, including oligomers (O), dimers (D) and monomers (M) from (A). (C) Analysis of SCD activity of the recombinant SCD enzymes used for Western blot analysis shown in (A). Data were collected from at least three independent experiments, and were expressed as means±S.E.M. Student's t test was performed to analyse differences in enzymatic activity between the DSP-treated and the non-cross-linked samples of each SCD enzymes, and significant differences (P<0.05) are indicated with an asterisk (*).