Figure 5

APOA4 activation of PI3K-AKT pathway is partly LRP1-dependent. (A) APOA4 promoted phosphorylation of AKT (pAKT) in a time-dependent manner. Representative western blot images are shown. After transfer of proteins, the membranes were cut into three sections before probing with specific antibodies for western blots. The top section (> 75kD) was used for LRP1 detection, the middle sections (50–75 kD) for pAKT and total AKT detection, and the bottom sections (25–50 kD) for GAPDH detection. This was necessary as stripping and re-probing compromises quantification and running separate gels would not allow for quantification. Bar graphs depicting normalized pAKT and AKT levels to GAPDH. Compared to 0 min, the pAKT/AKT ratio was significantly increased by 2.6-fold at 60 min. (B) Representative western blot images showing LRP1, pAKT, AKT, and GAPDH. After pAKT detection, the blot was stripped and used for detecting total AKT. At 60 min post APOA4 treatment, when LRP1 levels were reduced by ~ 50% compared with controls, the pAKT/AKT ratio decreased by ~ 58%, suggesting that APOA4-stimulated increases in pAKT are in part, LRP1-dependent (n = 3/group).