Experimental validation of computerised models of clustering of platelet glycoprotein receptors that signal via tandem SH2 domain proteins
Fig 8
The Syk inhibitor PRT-060318 inhibits rhodocytin-and AYP1-induced CLEC-2 clustering.
(A) Confocal microscope calibration using Atto-488 dye (10 nM) in water at 25°C. The axial (Zo) and lateral (Wo) radii were determined to be ~1.72+0.72 μm and ~0.23+0.03 μm respectively and the confocal volume was ~0.17+0.05 μm3. > 110 FCS calibration measurements were taken in five experiments. Data are presented as mean ± s.d. (n = 5). (B) (i) Representative confocal microscopy images showing membrane localisation of CLEC-2-eGFP in resting and stimulated cells (field of view = 52 x 52 μm) (scale bar = 5 μm). (Bii) Box plots showing the molecular brightness (cpm s-1) of CLEC-2-eGFP, centre lines represent median, box limits indicate the 25th and 75th percentiles, and whiskers extend to minimum and maximum points. Concentration of rhodocytin (30 nM), mAb AYP1 (6.6 nM), PRT (10 μM) were used. Significance was measured with Kruskal-Wallis with Dunn’s post-hoc where P ≤ 0.05. In (ii) # = significance compared to CLEC-2 alone. FCS measurements were taken in 35–48 cells; n = 3–5.