Extended Data Fig. 8: Epitope mapping of anti-human APRIL antibodies, and native canonical and non-canonical APRIL differ in size but are produced by the same gene.
From: APRIL limits atherosclerosis by binding to heparan sulfate proteoglycans
a–g, Epitope mapping of anti-human APRIL antibodies. a, The epitopes recognized by Aprily1, 2, 3, 5 and 10 were mapped by western blot of truncated APRIL proteins. b–d, Aprily5 and Aprily1 or Aprily2 recognize distinct epitopes (d), whereas Aprily3 and Aprily10 recognize epitopes distinct from those of Aprily1, Aprily2 and Aprily5 (e, f). g, Expression of all constructs was validated by western blot with anti-Fc antibody. h, i, Human serum was depleted of APRIL using the anti-human APRIL antibodies Aprily1, Aprily2, Aprily3, Aprily5, Aprily6, Aprily8, Aprily9, Aprily10, Mahya-1, 110.6, the biological atacicept (TACI-Ig: a recombinant fusion protein of the receptor TACI and the Fc region of Ig, that binds to APRIL) or the negative control EctoD1, and then analysed with a c-APRIL-specific (h; ELISA 1) or an nc-APRIL-specific ELISA (i; ELISA 2). Data are derived from one experiment in this format. j–n, Native canonical and non-canonical APRIL differ in size. j, Flag–human APRIL (from c-APRIL ELISA 1 standards) was depleted on TACI–Fc (or TNFR2–Fc as control) and/or on Aprily2 (or mIgG1 as control). APRIL was then detected by c-APRIL-specific (top) or nc-APRIL-specific (bottom) ELISA. k, l, Flag–human APRIL (from APRIL ELISA 1 standards) was depleted on immobilized TACI–Fc or on Aprily2, and the flow-through was then size-fractionated by size exclusion chromatography (SEC) and detected in fractions by c-APRIL-specific (k; ELISA 1) or nc-APRIL-specific (l; ELISA 2) ELISA. TACI–Fc and Aprily2 beads used for depletion were then acid-eluted. m, n, The neutralized eluate was size-fractionated, and APRIL in fractions was detected with c-APRIL-specific (m) or nc-APRIL-specific (n) ELISA. These results indicate that Flag–c-APRIL has the size of a 3-mer, whereas nc-APRIL is much larger. o, p, Canonical and non-canonical APRIL are produced by the same TNFSF13 gene locus. The TNFSF13 gene (which encodes APRIL) was inactivated in human macrophage cell line U937 by CRISPR–Cas9 technology. As a control, the TNFSF13B gene (which encodes BAFF) was also deleted. APRIL in supernatants was measured with a c-APRIL-specific (o) and an nc-APRIL-specific (p) ELISA. 105, 110, 301 and 302 depict different clones.
