|
| Figure 4: Screening analysis of PCSK9-mediated LDLR degradation by western blot. A. 6 positive compounds (compound A-F) were added to the culture medium individually with D374Y-PCSK9 to HepG2 cells. Reduction of endogenous mature LDLR (M) in HepG2 cells was analyzed by western blot. Compound B ( Figure 4, lane 4, Table 1, colchicine) has been detected to inhibit D374Y-mediated LDLR degradation, showing increased LDLR after treatment. Equal loadings were confirmed using anti tubulin antibody. B. Dose response of the compound B (colchicine) on inhibiting D374Y-mediated LDLR degradation in the western blot analysis. The ratio of LDLR/tubulin in untreated sample (Figure 4, lane 1) was assigned a value of 100%. The ratio of LDLR/ tubulin in D374Y-PCSK9 treated sample (13.4 nM, 16 h, Figure 4, lane 2) has a value of 25%. Four different concentrations of colchicine were used to calculate the IC50 of inhibition of PCSK9-D374Y by colchicine (n=3). The IC50 was 0.131 μΜ. |
