FE65 increases cell surface levels of VLDLR A. COS7 cells were transiently transfected with full length VLDLR for 24 hours. After transfection, cells were grown in serum-free media and incubated with MG132 (10 uM) (lane 2) or DMSO (lane 1) as control for 24 hr. Cell lysates (20 ug/lane) were then probed with anti-HA antibody. Consistent with previous findings, VLDLR CTFs were only detectable in the presence of MG132. B. COS7 cells were transiently transfected with VLDLR and empty vector or VLDLR and FE65. Secreted VLDLR was measured in conditioned media (14 ul) with 5F3 antibody. Total VLDLR and VLDLR CTF were measured in cell lysates (20 ug/lane) with anti-HA. FE65 increased levels of sVLDLR, suggesting that FE65 affects VLDLR processing. C. COS7 cells were transfected with VLDLR and vector (lanes 1, 3) or VLDLR and FE65-myc (lanes 2, 4). Cell surface proteins were biotin-labeled, isolated with avidin-beads, and immunoblotted with the 5F3 antibody for VLDLR detection. Full length FE65 increased surface levels of VLDLR (upper blot). Expression of VLDLR across all transfections in shown in the lower blot. D. Quantification of surface levels of VLDLR normalized to control in (C). FE65 increases cell surface levels of VLDLR by 118% (n = 4, p < 0.001). E. Cultured hippocampal neurons (DIV14) were transfected with GFP, VLDLR, and empty vector (upper panel) or GFP, VLDLR, and FE65 (lower panel). Surface VLDLR was measured with the 5F3 antibody by immunofluorescence of live cells. F. Quantification of cell surface VLDLR intensity in neuronal processes in (E). The cell surface staining in neuronal processes showed a 1.2 fold increase in cell surface levels of VLDLR with FE65 (n = 10, p < 0.05). G. Cultured hippocampal neurons (DIV14) were transfected with GFP, VLDLR, and empty vector (upper panel) or GFP, VLDLR, and FE65 (lower panel). Total levels of VLDLR were measured with the 5F3 antibody by immunofluorescence after permeabilization of cells. H. Quantification of total VLDLR intensity in neuronal processes in (G).