Fig. 1. MMP-13 binds to PD-1H expressed on OCLs.

A For MMP-13 pull-down, BSA or MMP-13-His₆ recombinant protein were incubated with M-CSF primed mouse non-adherent mononuclear bone marrow cell lysates, followed by Ni-NTA agarose beads affinity pull-down. MMP-13 binding proteins were extracted and analyzed by LC/MS/MS Mass spectrum. Figure was created with BioRender.com. B Among all the proteins, PD-1H showed the highest relative abundance in the MMP-13 pull-down samples compared to BSA control samples (n = 3, each sample was analyzed twice). P-values were determined by two-sided Student’s t-test. C HEK293 cells (not expressing MMP-13) were transfected with Myc tagged MMP-13 alone or together with Flag-PD-1H, followed by IP using Flag antibody. Whole-cell lysates (WCL) were analyzed as input. The experiment was performed three times with similar observations. D HEK293 cells were transfected by empty vector (EV) or Flag-PD-1H, followed by incubation with MMP-13-GFP fusion protein. Cells were stained with FITC-anti-GFP, PE-anti-PD-1H and nuclei by DAPI. Scale bar, 10 μm. The experiment was performed three times with similar observations. E Mouse OCL cell lysates were incubated with RPMI8266 conditioned serum free medium, followed by IP using anti-PD-1H antibody. Rabbit IgG was used as control. The experiment was performed three times with similar observations. F HEK293 cells were transfected with Flag-PD-1H alone or together with Myc tagged full-length MMP-13 (FL), E223A enzymatic dead mutant, pro-catalytic domain (Cat, aa 1–267) or Hemopexin domain (Hem, deletion aa 37–267), followed by IP using anti-Myc (9E10) antibody. WCL were analyzed as input. The experiment was performed three times with similar observations. G HEK293 cells were transfected with MMP-13 alone or together with Flag-PD-1H full-length (FL) or extracellular domain (ECD), followed by IP using Flag antibody. Whole-cell lysates were analyzed as input. The experiment was performed three times with similar observations. H Purified PD-1H-ECD-Fc recombinant protein was incubated with purified MMP-13 WT-Myc or E223A-Myc recombinant protein and pulled down by Myc magnetic beads. The experiment was performed three times with similar observations.