FIGURE 6.

Identification of functional interaction of SORT1 and CES1 in HepG2 cells and mouse livers. A, HepG2 cells were infected with Ad-null or Ad-SORT1-FLAG at an MOI of 1 for 16 h. Cell lysate was used for immunoprecipitation with anti-FLAG antibody conjugated to magnetic beads. Immunoprecipitates were subjected to SDS-PAGE and silver staining before being submitted for proteomics analysis. NC, negative control. B, HepG2 cells were infected with Ad-null or Ad-SORT1-FLAG at an MOI of 1 for 16 h. Cell lysate was used for immunoprecipitation with anti-FLAG antibody conjugated to magnetic beads. SORT1-FLAG was detected with anti-FLAG antibody. Non-infected HepG2 cell lysate was used to detect CES1 band as controls. This experiment was performed three times, and all experiments showed CES1 co-precipitation with SORT1. C, validation of CES1 and CES3 antibodies with liver lysate from Ces1 and Ces3 knock-out mice. TGH, triacylglycerol hydrolase. D, immunoprecipitation of SORT1 from mouse livers expressing SORT1-FLAG followed by Western blotting analysis. Mice were injected with Ad-null. This experiment was repeated three times, and CES1 was co-precipitated with SORT1 in all experiments. E and F, HepG2 cells were infected with Ad-shSORT1 at an MOI of 5 or 10 for 24 h. Ad-scramble was used to infect control cells. In E, Western blotting was performed to detect SORT1 and CES1 protein. Average band intensity of 3 independent experiments (normalized to actin) was shown as values below the blot. In F, real-time PCR was used to measure CES1 and SORT1 mRNA in three experiments. Results are expressed as mean ± S.D. * indicates statistical significance, versus Ad-siCon. G and H, liver protein and mRNA of WT and Sort1 KO mice on chow diet. Values below the blot indicate the relative band intensity (normalized to actin) in each group. Results are expressed as mean ± S.E. * indicates statistical significance, versus WT mice. n = 6.