Figure 3
Genetic knockdown of BMP1 decreases the proteolytic cleavage of LDLR. (A) Immunoblot analysis of LDLR in cell lysates and conditioned media from HepG2 cells treated either with siRNA against BMP1 (Dharmacon) or with a non-targeting control siRNA. Blot images were cropped to highlight the FL and CTF bands in cell lysates using the AF2148 antibody and the FL and NTF bands in cell lysates and conditioned media, respectively, using the Ab14056 antibody. Full blot images are presented in the Supplementary western blot dataset. (B) Densitometric analysis of the NTF from (A). Data expressed as percentage of control and shown as mean ± SEM, statistical analysis performed using a Student’s T-test ***p < 0.001, n = 6. (C) Immunoblot analysis of LDLR in cell lysates and conditioned media from HepG2 cells treated either with siRNA against BMP1 (Ambion) or with a non-targeting control siRNA. Blot images were cropped to highlight the FL and CTF bands in cell lysates using the AF2148 antibody and the FL and NTF bands in cell lysates and conditioned media, respectively, using the Ab14056 antibody. Full blot images are presented in the Supplementary western blot dataset. (D) Relative expression of BMP1 mRNA in HepG2 cells treated with siRNA against BMP1 (Ambion) or with a non-targeting control. Data expressed relative to control and shown as mean ± SEM, statistical analysis performed using an independent t-test with Welch’s correction for two sample comparison **p < 0.01, n = 3. (E) Immunoblot analysis of LDLR and BMP1 in cell lysates from HepG2 cells treated with siRNA against BMP1 (Ambion) or with a non-targeting control siRNA and then transfected either with an empty vector, BMP1-FLAG or BMP1 E214Q-FLAG constructs. Blot images were cropped to highlight the FL and CTF band of LDLR using the AF2148 antibody and BMP1 using the AF1927 antibody. Full blot images are presented in the Supplementary western blot dataset. (F) Densitometric analysis of FL LDLR and the CTF from (E). Data expressed as percentage of NT siRNA control (not shown in graph) and shown as mean ± SEM, statistical analysis performed using an ANOVA with Bonferoni post-hoc pairwise analysis **p < 0.01, ***p < 0.001, n = 3.
