Figure 1

Human LDLR is proteolytically cleaved in its extracellular ligand binding domain by BMP1. (A) Schematic of the domain organisation of LDLR with a C-terminal FLAG tag showing the epitopes detected by the antibodies used in the study, antibody AF2148 (R&D Systems) raised against the entire ectodomain of LDLR, antibody Ab14056 (Abcam) raised against a recombinant protein fragment corresponding to amino acids 29–205 of LDLR) and antibody α-FLAG (Sigma-Aldrich) the anti-FLAG M2 antibody. TM, transmembrane domain; EGF, epidermal growth factor-like domain; F, FLAG epitope. (B) Immunoblot analysis with the indicated antibody of lysates and conditioned media samples from HepG2 cells expressing full-length FLAG-tagged human LDLR. Bands of interest were cropped from western blots of either media or lysate samples using each of the three antibodies. Images from separate western blots were combined but are separated by the dashed black line. Full blot images are presented in the Supplementary western blot dataset. (C) Schematic showing the proposed cleavage of the 160 kDa full-length (FL) LDLR to generate the 36–40 kDa NTF and 120 kDa CTF. (D) Immunoblot analysis of LDLR (antibody AF2148) without and with deglycosylation in liver biopsy samples from three separate individuals. The blot image was cropped to highlight the FL and CTF bands, full blot images are presented in the Supplementary western blot dataset. (E) Immunoblot analysis following incubation of rhLDLR (500 ng) with increasing amounts of rBMP1 at 37 °C for 1 h. (F) Immunoblot analysis following incubation of rhLDLR (500 ng) with rBMP1 (12.5 ng) in the absence or presence of the BMP1 inhibitor UK383367 (10 μM) at 37 °C for 1 h. (G) Immunoblot analysis following pre-incubation of rhLDLR (500 ng) in the absence or presence of LDL (5 µg), RAP (7.14pmol) or UK383367 (10 μM) for 30 min on ice followed by the addition of 12.5 ng rBMP1 and further incubation at 37 °C for 1 h. (H) Densitometric analysis of the Ab14056 immunoblot from (C) to determine the amount of FL and NTF as a percentage of total LDLR, data shown as mean ± SEM, statistical analysis using ANOVA with Tukey post-hoc pairwise analysis *p < 0.05, n = 3. For panels E–G, blot images were cropped to highlight the FL and CTF bands using the AF2148 antibody and the FL and NTF bands using the Ab14056 antibody due to different exposure times for visualisation of the FL and NTF bands. Full blot images are presented in the Supplementary western blot dataset.