Membrane expression and functionality of hLDLR expressed by the CHO-hLDLR-EGFP cell line.

<p>(A) Representative confocal photomicrographs of immunostained CHO-hTfR-EGFP (negative control) and CHO-hLDLR-EGFP cells (green) with an anti-LDLR antibody diluted at 1/100 (red) following triton X100 permeabilization of the cell membrane. Cell nuclei are labeled with Hoechst#33258 at 0.5 μg/mL (blue). Co-labeling appears in yellow in the merged pictures. Only the cells with stable expression of the hLDLR-EGFP construct express the membrane receptor. (B) Representative confocal photomicrographs of CHO-hTfR-EGFP and CHO-hLDLR-EGFP cells (green) after a 10 min incubation period with DiI-LDL 15 μg/mL (red). Cell nuclei are labeled with Hoechst#33258 (blue). Co-labeling appears in yellow in the merged pictures. DiI-LDL is essentially bound to the CHO-hLDLR-EGFP cells indicating that the hLDLR-EGFP chimera receptor binds its ligand. (C) Western blots performed on cell membrane preparations of CHO cells expressing hLDLR-EGFP, rLDLR-EGFP and mLDLR-EGFP compared to CHO WT, using anti-hLDLR (1/800) and anti-rat and mouse LDLR antibodies (1/1000). These bands are also labeled with an anti-EGFP antibody. Bands of 140 kDa and 190 kDa correspond respectively to the immature and mature LDLR-EGFP fusion proteins (arrows). Actin was used to check loading of equal amounts of protein.