Figure 2
Cleavage of human LDLR by BMP1 reduces the binding and cellular uptake of LDL. (A) Immunoblot analysis following incubation of rhLDLR-His (5 μg) with rBMP1 (200 ng) at 37 °C for 1 h. The blot image was cropped to highlight the FL and CTF bands, full blot images are presented in the Supplementary western blot dataset. (B) Dot blot of BODIPY®-LDL (1 μg/ml) binding to rhLDLR-His digested with BMP1 (from A), BACE1-His and PCSK9-FLAG (all 0.5 μg). Membranes were re-probed with anti-His and anti-FLAG antibodies. (C) Densitometric analysis of the BODIPY-LDL dot blot from (B) showing the percentage binding to rhLDLR-His in the absence or presence of rBMP1. Data expressed as percentage of control and shown as mean ± SEM, statistical analysis performed using a Student’s T-test ***p < 0.001, n = 4. (D) Immunoblot analysis of LDLR with actin as a loading control in lysates from CHO-A7 cells expressing either full-length (FL) LDLR or 120 kDa CTF, or the empty vector (EV). The blot images were cropped to highlight the FL and CTF bands using the AF2148 and anti-FLAG antibodies and actin using the AC15 antibody. The full blot images are presented in the Supplementary western blot dataset. (E) Immunoblot analysis of LDLR from lysates (L) and from the cell-surface fraction (S) in CHO-A7 cells expressing either FL LDLR or 120 kDa CTF followed by incubation with either vehicle (DMSO) or EZ-Link™ Sulfo-NHS-SS-Biotin (0.5 mg/ml) for 20 min on ice and immunoprecipitation with Streptavidin-agarose beads for 3 h at 4 °C. The blot image was cropped to highlight the FL and CTF bands using the AF2148 antibody. The full blot image is presented in the Supplementary western blot dataset. (F) Immunofluorescence microscopy images showing LDLR expression (green) and Dil-LDL binding (red) in CHO-A7 cells expressing either FL LDLR or 120 kDa CTF followed by incubation with Dil-LDL for 30 s on ice. (G) Quantification of Dil-LDL mean intensity in either the FL LDLR or 120 kDa CTF expressing cells from (F), data shown as mean ± SEM, statistical analysis performed using a Student’s T-test *p < 0.05, n = 7. (H) BODIPY®-LDL uptake in CHO-A7 cells expressing either FL LDLR or 120 kDa CTF. Data expressed as percentage of FL following subtraction of empty vector uptake as a baseline and shown as mean ± SEM, statistical analysis performed using a Student’s T-test ***p < 0.001, *p < 0.05, n = 4.
