Figure 3

APOA4 and LRP1 interaction increased concomitantly with lipid feeding. (A) APOA4 interacted with LRP1 in male (left) and female (right) WT gonadal fat tissues collected 5-h after fasting, as revealed in co-immunoprecipitation assay; pull down with non-specific IgG did not detect LRP1 or APOA4. Representative gel lanes are shown (n = 3). (B) Overnight-fasted male mice were gavaged with a single dose of lipid. Epididymal fat tissues were collected before and at 15, 30, 60, 120 min after lipid-gavage and used for co-immunoprecipitation. Triglyceride (TG) levels increased at 15 min by 20% and peaked at 60 min by 44% after lipid gavage, compared to fasted state (0 min). Plasma APOA4 levels also increased by 1.8-fold at 15 min and remained elevated even at 120 min post lipid feeding. APOA4 interaction with LRP1 was enhanced at 30 and 60 min when plasma TG and APOA4 levels were also increased; but no interaction was observed with IgG pull-downs (n = 3). After transfer of proteins, the membranes were cut into two or three sections before probing with specific antibodies for western blots shown. The top section (> 75kD) was used for LRP1 detection, the bottom sections (25–75 kD) for APOA4 detection. This was necessary as stripping and re-probing compromises quantification and running separate gels would not allow for quantification. (C) Representative immunofluorescence confocal images of APOA4- and LRP1-immunoreactivity (IR) in epididymal fat tissue sections prepared from male WT mice fasted for 5 h. Robust co-localization (Merge, yellow) of LRP1 (Green) with ApoA4 (Red) was observed in epididymal fat tissue (n = 2 technical and 3 biological replicates). Scale bars: 50 μm and 10 µm.