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. Author manuscript; available in PMC: 2024 May 2.
Published in final edited form as: Cell Rep. 2023 Jan 5;42(1):111930. doi: 10.1016/j.celrep.2022.111930

Figure 4. CXCL4 oligomerisation drives leukocyte recruitment.

Figure 4.

(A) AUC analysis of CXCL4 and the mutant K50E to show sedimentation coefficients (indicative of size). (B) Schematic of oligomerisation states of CXCL4 or the CXCL4 K50E mutant. (C) BLI analysis of CXCL4 K50E binding to immobilised heparin dp8. (D) In vivo leukocyte (CD45+) recruitment (air pouch) to CXCL4 K50E mutant. (E) Schematic of RTO antibody binding to CXCL4 monomer to inhibit oligomerisation. (F) CXCL4 (50 nM) binding to dp8 was monitored in the absence and presence (at a range of concentrations) of RTO antibody. (G) Final CXCL4:dp8 signal (after washing) from (F) is plotted with and without pre-incubation with RTO antibody (25 nM). (H) Schematic of FRAP assay to analyse HS cross-linking. (I) FRAP analysis of GAG in-plane mobility with CXCL4 alone or in combination with the RTO antibody, images post bleaching at indicated times. (J) Fluorescence recovery over time. (K) CXCL4 mediated leukocyte recruitment (CD45+) with and without RTO antibody.

D, G, J and K are mean with 95% confidence intervals and represent at least two separate experiments where data have been pooled. Each dot in D and K represents an individual mouse and each dot in G represents a technical replicate. A, C, F, I and J are representative of at least two separate experiments. Data in D and K are normalised to vehicle controls. Individual p values are shown, G analysed using an unpaired t-test and K analysed using a one-way ANOVA with a post-hoc Tukey analysis.