Fig 2: The LA domain of LDLR is responsible for the uptake of Tcnα.a Schematic drawing of Ldlr, Ldlr∆LA, Ldlr∆LB, and Ldlr∆NPxY. b The HeLa WT or LDLR‒/‒ cells were transfected with mock, Ldlr, Ldlr∆LA, Ldlr∆LB, and Ldlr∆NPxY, followed by incubating with Tcnα for 3 h. The percentages of round-shaped cells are plotted on the chart. The blue dash line indicates 6 nM. Error bars (n = 6) indicate mean ± SD. c The indicated HeLa cells were incubated with Tcnα (6 nM, 3 h) and the images were captured. Red fluorescence (mCherry) marked transfected cells. The scale bar represents 50 μm. d The round-shaped cells among all mCherry-positive cells shown in c were quantified and plotted in a bar chart. Error bars (n = 6) indicate mean ± SD, *P < 0.05, ***P < 0.001, n.s. = not significant, two-sided Student’s t-test.

Fig 3: Increased expression of LDLr and LRP1. (A) RT-QPCR analyses of mRNA expression of LDLr, VLDLr and SR-B1, n = 4 per group. (B) mRNA expression of VLDLr in HL-1 cells incubated in hypoxia for 0.5, 1, 6 and 24 h (n = 3). (C) Representative immunoblots of LDLr and LRP1. (D–E) Quantification of LDLr protein bands (D) and LRP1 protein bands (E) n = 6–7 per group. Results are shown as mean ± SEM, *P<0.05 vs. control, **P<0.01 vs. control, ***P<0.001 vs. control.

Fig 4: Selective anti-ALK mAb2 attenuates the progression of atherosclerosis in mice and mAb2 operates independently of LDL receptor in vivo.a-d, Analysis of weight (a), plasma total cholesterol (b), triglyceride (c), and blood glucose level (d) from Ldlr−/− mice fed a western diet for 12 weeks with IgG or mAb2 treatment. Bi-weekly injections of 250 μg per injection. n = 12. P = 0.0912 for a, P = 0.5239 for b, P = 0.8829 for c, and P = 0.9913 for d. Values show mean ± S.E.M. P values were calculated by one-way ANOVA with Sidak’s multiple comparisons test. e,f, Relative mRNA (e) and protein LDLR (f) expression level from the livers of LDLR WT, KO, and Het mice fed a Western diet for 6 weeks with antibody treatment. n = 5. P < 0.0001 for e. Values show mean ± S.E.M. P values were calculated by one-way ANOVA with Sidak’s multiple comparisons test. g, Relative sterol-sensing gene, SREBF2, expression from the livers of LDLR WT, KO, and Het mice fed a Western diet for 6 weeks with antibody treatment. n = 3. P = 0.7954. Values show mean ± S.E.M. P values were calculated by two-tailed unpaired t-test. Source Data.

Fig 5: mAb2 concentration is reduced to half in 4 days and does not inhibit BMP-9 signaling or vascular tone in vivo.a,b, Representative en face images (a) and quantification (b) of DiI-LDL in Ldlr−/− mouse aorta injected with either IgG or increasing doses of mAb2 (100-500 μg/mouse). One image was counted per mouse in five mice per group for a total of n = 5. Scale bar: 20μm. Image J software was used to quantify the samples. P < 0.0001 for b. Values show mean ± S.E.M. P values were calculated by one-way ANOVA with Sidak’s multiple comparisons test. c,d, Representative en face immunofluorescence images (c) and quantification (d) of endogenous p-SMAD1/5 localization in aortic arch of Ldlr−/− mice injected with PBS, IgG, or mAb2. P = 0.8607 for d. Values show mean ± S.E.M. P values were calculated by one-way ANOVA with Sidak’s multiple comparisons test. Scale bar: 20μm. e, Serum antibody concentrations were measured using an ELISA assay. Anti-ALK mAb concentration change over seven days after single injection (250 μg) to Ldlr−/− mice fed a Western diet for 7 days. Values show mean ± S.E.M. f-h, Representative confocal images (f), and analysis of ALK1 (g) and VE-cad intensity (h). Two images were counted per mouse in three mice per group for a total of n = 6. Scale bar: 20μm. P = 0.7636 for g and P = 0.7679 for h. Values show mean ± S.E.M. P values were calculated by two-tailed unpaired t-test. Image J software was used to quantify the ALK1 and VE-cad intensity. i-k, Relative VE-cad (i), PECAM-1 (j), and ALK1 (k) expression from isolated aortic ECs of Ldlr−/− mice fed a Western diet for 6 weeks with IgG or mAb2 treatment. n = 3. P = 0.8225 for i, P = 0.2299 for j, and P = 0.9459 for k. Values show mean ± S.E.M. P values were calculated by two-tailed unpaired t-test. l, Isolectin B4 staining of retinal vessels of Ldlr−/− mice fed a Western diet for 12 weeks with either IgG or mAb2 treatment. n = 6. Source data