Extended Data Fig. 4: APRIL binds HSPG2.

a, Representative photomicrographs (left) and quantification (right) of HUVECs incubated with either human or mouse Flag–APRIL in the presence or absence of heparin and stained with the anti-Flag M2 antibody conjugated to FITC and analysed by confocal microscopy. b, Flow cytometry analysis of HUVECs incubated with Flag–APRIL or flag-tagged bacterial alkaline phosphatase (Flag–BAP) and stained with the anti-Flag M2 antibody. c, Identification of protein binding partners of APRIL in HUVEC culture by performing a pull-down assay with agarose beads coupled to the anti-Flag M2 antibody followed by MS analysis. d, APRIL binding to coated HSPGs from mouse basement membrane quantified by ELISA determined in triplicate (****P < 0.0001, two-tailed unpaired Student t test). Data shown are representative of at least two independent experiments (a, b, d). e, f, Photomicrographs of mouse carotid artery sections incubated with mouse multimeric Flag–APRIL and stained with either an anti-Flag antibody conjugated to PE or with an anti-mouse APRIL biotinylated antibody (2C8) and streptavidin conjugated to PE (e; scale bar, 75 μm, data derived from two independent experiments) or with mouse multimeric Flag–APRIL in the presence or absence of heparin and stained with an anti-APRIL biotinylated antibody (2C8) or with an anti-HSPG2 or only secondary antibody (only 2nd) (f; scale bar, 75 μm, data derived from one experiment). g, Quantitative surface plasmon resonance (Biacore) analysis of the affinity of soluble human Fc–APRIL (total), human canonical Fc–APRIL (human Fc–c-APRIL), human non-canonical Fc–APRIL (human Fc–nc-APRIL), mouse canonical Fc–APRIL (mouse Fc–c-APRIL), mouse non-canonical Fc–APRIL (mouse Fc–nc-APRIL) and negative controls EDAR–Fc and human Fas–Fc to biotinylated heparin coupled to streptavidin Sensor Chip A (n = 3 independent experiments). All results show mean ± s.e.m. IntDen, integrated density.