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. 2021 Oct 20;297(5):101315. doi: 10.1016/j.jbc.2021.101315

Figure 5.

Figure 5

PLA signals of vWF-CD63 and vWF-P-selectin in HUVECs. A, control groups: (a) reagent-negative control where normal mouse and rabbit IgGs were used as primary Abs, (b) negative control where mouse anti-vWF Abs and rabbit anti-Rab5 Abs were used as primary Abs, (c) mouse anti-vWF Abs alone, (d) rabbit anti-CD63 Abs alone, (e) rabbit anti-P-selectin Abs alone, (f) no first Abs, and (g) positive control where mouse anti-talin Abs and rabbit anti-α-catenin were used as primary Abs (red). B and C, IFA staining: the representative PLA signals (red) of vWF-P-selectin (B, arrowheads) and vWF-CD63 (C), respectively, in HUVECs. (a) vehicle only, (b) rTNFα, (c) NY173 pretreatment + rTNFα, and (d) I942 pretreatment + rTNFα. The nuclei in panels A–C were counterstained with DAPI (blue). The scale bars indicate 20 μm. The graphs show the quantitative analysis of vWF-P-selectin PLA signals (B) and vWF-CD63 PLA signals (C) using ImageJ software. Five 40× microscopic fields were examined for each case. The results are expressed as dot signals enumerated in each 40× field. n  = 5 per group. ∗p < 0.05. Abs, antibodies; DAPI, 4′,6-diamidino-2-phenylindole; HUVECs, human umbilical vein endothelial cells; IFA, immunofluorescence assay; IgG, immunoglobulin G; NS, not significant; PLA, proximity ligation assay; rTNFα, recombinant tumor necrosis factor-α; vWF, von Willebrand factor.