Journal: Bone research
Article Title: Skeletal progenitor LRP1 deficiency causes severe and persistent skeletal defects with Wnt pathway dysregulation.
doi: 10.1038/s41413-024-00393-x
Figure Lengend Snippet: Fig. 6 LRP1 mediates endocytosis of Wnt5a, a core non-canonical WNT/planar cell polarity (PCP) pathway component. a Schematic diagram illustrating the long bone phenotype of Lrp1flox/flox/Prrx1Cre mice. Full-length sLRP1 was coated onto microtiter plates and the binding of 0–200 nmol/L Wnt5a (b), Wnt11 (c), Wnt3a (d) was measured using specific antibody for each Wnt as described under “Materials and Methods”. Mean values of technical duplicates for none-coating, LRP1-coating and after normalisation were shown as circles, squares and triangles, respectively. Extrapolated KD,app values were estimated based on one-phase decay nonlinear fit analysis (black lines). WT and LRP1 KO MEFs (n = 3) were incubated with 40 nmol/L Wnt5a for 0.5–60 min and Wnt5a in the cell lysate was detected by Western blotting (e). The relative amount of Wnt5a was expressed by taking the amount of Wnt5a after 60-min incubation as 1 (f). Circles represent individual mice and bars show the mean ± Svalues for the amount of Wnt5a after incubation for 5–30 min in WT versus LRP1 KO MEFs were evaluated by two-way ANOVA. *P < 0.05. Representative images of confocal microscopy analysis for Wnt5a and LRP1 in WT and LRP1 KO MEFs (n = 3) (e) or human normal chondrocytes (n = 3) f Cells were incubated with 20 nmol/L Wnt5a for 3 h in the absence (g, h) or presence of 500 nmol/L RAP (h). Wnt5a, LRP1, cytoskeleton and nucleus were visualised as described under “Materials and Methods”. Scale bar, 10 µm. Regions delineated by the white squares in the panels have been magnified in the top right (g). WT and LRP1 KO MEFs (n = 3) were incubated with 20 nmol/L Wnt5a for 3–24 h and Wnt5a in the medium and cell lysate were detected by Western blotting (i). Densitometric analysis of immunoreactive Wnt5a bands was carried out. The relative amount of Wnt5a in the media, cell lysate and both media and cell lysate (total) were expressed by taking the amount of Wn5a after 3-h incubation in WT MEFs as 1 (j). k human normal chondrocytes (n = 3) were incubated with 20 nmol/L Wnt5a for 1–24 h and analysed as in a and b. The relative amount of Wnt5a after 24-h incubation was expressed by taking the amount of Wn5a after 1-h incubation as 1. Circles represent individual experiment and bars show the mean ± SD
Article Snippet: Each sample was then incubated with anti-Wnt5a antibody (AF645, R&D systems) and anti-LRP1 antibody (ab92544, Abcam) for overnight at 4 °C.
Techniques: Binding Assay, Incubation, Western Blot, Confocal Microscopy
R&D Systems is a verified supplier 
R&D Systems manufactures this product 
