Figure 6

Downregulation of SNX12 accelerates the endocytosis of BACE1 and decreases the cell surface level of BACE1. (A) After RNAi downregulation of SNX12, SH-SY5Y cells were subjected to biotinylation. Cell lysates were affinity-precipitated with streptavidin-agarose beads to pull down biotinylated proteins that were at the cell surface. The levels of biotinylated BACE1 and APP, as well as their total protein levels, were analyzed by Western blot. Cell surface levels of BACE1 were quantified by densitometry and normalized to that of control (set as one arbitrary unit) for comparison. N = 3, *: p < 0.05. (B) Upon downregulation of SNX12 by RNAi, protein at the cell surface of SH-SY5Y cells was labeled with EZ-Link Sulfo-SS-Biotin. After chasing at 37 °C for the indicated times, cells were treated with glutathione at 4 °C to cleave biotin from remaining cell surface proteins. Cell lysates were affinity-precipitated with streptavidin-agarose beads. The levels of biotinylated (i.e. endocytosed) BACE1 were analyzed by Western blot. The levels of endocytotic BACE1 were quantified by densitometry and normalized to those of five percent of total (T) BACE1 (before chasing) for comparison. N = 3, *: p < 0.05.