Figure 2.

Effect of siRNA knockdown and plasmid overexpression of ADAM10 and ADAM17 on VLDLR shedding. Primary human RPE cells were transiently transfected with indicated siRNA (50 nM) or plasmid (1 μg/ml) for 24 h. Cells were subsequently infected with Ad-VLDLRII (MOI = 25) for 24 h. Then, culture media were replaced with serum-free DMEM for another 24 h. Finally, culture media and cell lysates were collected for Western blot analysis. A, representative images of Western blotting for sVLDLR in culture media and full-length VLDLR, ADAM10, and ADAM17 in cell lysates. The RPE cells were transfected with the siRNA for ADAM10, ADAM17, or control siRNA. B, quantification of densitometry of sVLDLR in culture media normalized by VLDLR in cell lysates in (A) (n = 3). C, representative images of Western blotting for sVLDLR in culture media, and VLDLR, ADAM10, and ADAM17 in cells transfected with plasmid overexpressing human ADAM10, human ADAM17, or RFP as control. D, protein levels of sVLDLR in the media in (C) were quantified and normalized by the full-length VLDLR in cell lysates (n = 3). E, representative images of Western blotting of sVLDLR in culture media, VLDLR, ADAM10, and ADAM17 in human primary RPE cells, which were transfected with siRNA knocking down human ADAM17 (siADAM17) or plasmid overexpressing human ADAM17 (pADAM17). F, quantification of densitometry of sVLDLR in culture media normalized by VLDLR in cell lysates in (E) (n = 3). In A, C, and E, p and m indicated precursor and mature forms of ADAM10 and ADAM17, respectively. Data were presented as mean ± SD. ∗∗p < 0.01. ADAM10, a disintegrin and metalloprotease 10; ADAM17, a disintegrin and metalloprotease 17; DMEM, Dulbecco's modified Eagle's medium; MOI, multiplicity of infection; RFP, red fluorescent protein; RPE, retinal pigment epithelium; sVLDLR, soluble ectodomain of VLDLR; VLDLR, very low-density lipoprotein receptor; VLDLRII, VLDLR variant II.