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. 2023 Jul 17;14:4271. doi: 10.1038/s41467-023-39769-8

Fig. 5. PD-1H binds to c-Src and mediates MMP-13 induced OCL activation.

Fig. 5

A WT OCLs differentiated without or with MMP-13 were stained by anti-c-Src (green), anti-PD-1H (red) and Acti-stain 670 phalloidin (gray) and DAPI (blue) and analyzed by confocal microscopy. Scale bar, 100 μm. The experiment was performed three times with similar observations. B–D c-Src was co-transfected in HEK-293 cells without or with Flag-PD-1H followed by co-IP by anti-Flag antibody (B), without or with Flag-PD-1H FL or extracellular domain (aa1-215) followed by co-IP by anti-Flag antibody (C), with Flag-PD-1H FL or △281-308 or R86A/F94A/Q95A triple point mutation (RFQ/AAA) followed by co-IP by anti-Flag antibody (D). The experiments were performed three times with similar observations. E WT or Pd-1h-/- OCLs differentiated without or with MMP-13 were stained with anti-c-Src (green), anti-PD-1H (red), Acti-stain 670 phalloidin (gray) and DAPI (blue). Images were obtained by confocal microscopy. Scale bar, 100 μm. C-Src/F-actin co-localization cell numbers per well were calculated (n = 3 wells per group). **P ≤ 0.01 by one-way ANOVA. F RAW264.7 cells were differentiated into OCLs without or with MMP-13 followed by co-IP using IgG control or anti-PD-1H antibody. The experiment was performed three times with similar observations. G BMMNCs from WT or Pd-1h-/- mice were cultured in OCL differentiation medium without or with MMP-13. Cell lysates were analyzed by western blotting with β-actin as loading control. Bands densitometry intensities from repeated experiments are shown. The experiment was performed three times with similar observations.