Fig 2: Absence of connective tissue MCs after selective deletion of SNAP23. Color legend on top applies to all graphs. A, representative images of ear sections stained with FITC-avidin (green) and Hoechst 3342 (blue); autofluorescence in the red channel delimited the dermis as the area between the epidermis and the auricular cartilage (dotted line). Scale bar = 100 μm. B, quantification of MCs (Hoechst+ nucleus surrounded by FITC+ granules) per area or dermis. n = 9. C, representative cytospins of peritoneal lavages stained with Wright-Giemsa. Arrowhead, example of a metachromatic MC. Scale bar = 100 μm. D, number of MCs (large cells with metachromatic granules) per volume of lavage. n = 10. E, representative scatter plot of flow cytometry of peritoneal lavage cells labeled with fluorescent antibodies against Kit/CD117 and FcεRIα. Inset square, double-positive cells. F, average fraction of MCs (double-positive cells) in the lavages. n = 10. White line, mean; box, 25th to 75th percentile; whiskers, 5th to 95th percentile. ∗∗∗p ≤ 0.001; all compared to F/F.

Fig 3: Abnormal platelet exocytosis in the absence of SNAP23. Color legend on top applies to all graphs. A, whole-blood samples were exposed to thrombin (Thr; 0.1 U/ml), collagen (10 μg/ml), or ionomycin (10 μM), and dense granule secretion was assessed by increase in extracellular ATP (luminometry). +/+, F/F and pΔ/+ samples were diluted (∼1:2.5) to match the platelet count in pΔ/Δ samples; an undiluted +/+ sample was used as a control. n = 7. B and C, washed platelets labeled with fluorescent antibodies against P-selectin and LAMP-1 studied under flow cytometry before and after stimulation with thrombin with or without ADP (10 μM), while recording the difference in mean fluorescence intensity (ΔMFI). Alpha and lysosomal granule secretion were assessed by increase in platelet surface translocation of P-selectin (B; n = 7) or LAMP-1 (C; n = 10), respectively. D, representative EM profiles of platelets before and after activation with thrombin. Scale bar = 1 μm. E, surface density (Sv) of platelet EM profiles before and after activation. n = 12; applies to E–G. F and G, volume density (Vv) of dense and alpha granules, respectively, before and after activation. White line, mean; box, 25th to 75th percentile; whiskers, 5th to 95th percentile. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001; all compared to F/F unless otherwise specified (horizontal lines).

Fig 4: Abnormal number, expression of surface proteins, and activation of platelets in the absence of SNAP23. Color legend on top applies to all graphs. Whole-blood samples were loaded with a predetermined number of fluorescent beads, labeled with fluorescent antibodies against CD41 and P-selectin, and studied under flow cytometry. A, representative scattergrams of samples from SNAP23F/F and SNAP23pΔ/Δ littermates. FSC, forward scatter; SSC, side scatter; orange dots, CD41+ particles. B, representative histograms comparing CD41 fluorescence and FSC in the same F/F and pΔ/Δ samples. C, concentration of platelets (CD41+ particles) in whole-blood samples using fluorescent beads to calculate the original volume. n = 7; applies to C–F. D, difference in particle sizes estimated using the mean FSC values of CD41+ particles. E, mean fluorescence intensity (MFI) for CD41 in CD41+ particles. F, MFI for P-selectin in CD41+ particles. G, MFI for PAR4 in fixed and permeabilized platelets (total) or intact platelets (surface). n = 11. H, peak and area under the curve (AUC) over time of intracellular calcium concentrations ([Ca2+]i) measured by ratiometry in Fura-2 AM-loaded platelets activated with thrombin (0.1 U/ml). n = 12. I, MFI of intact platelets labeled with an antibody (Jon/A) against the activated conformation of αIIbβ3 before and after exposure to thrombin (Thr; 0.1 U/ml) with and without ADP (10 μM). n = 7. White line, mean; box, 25th to 75th percentile; whiskers, 5th to 95th percentile. ∗p ≤ 0.05, ∗∗p ≤ 0.01; all compared to F/F.

Fig 5: Direct phosphorylation of recombinant SNAP-23 (rSNAP-23) at Ser95 by recombinant PKM2 (rPKM2).(a) Direct phosphorylation of rSNAP-23 by rPKM2. The rSNAP-23 was incubated with or without PEP, rPKM2 or PEP plus rPKM2 at room temperature for 1 h. The reaction mixtures were then subjected to SDS-PAGE or Phos-tag SDS-PAGE analysis. SNAP-23 was detected by anti-SNAP-23 antibody in WB analysis. (b) Phosphorylated SNAP-23 by rPKM2 analysed by mass spectrometry (MS). Note that MS analysis of tryptic fragment of rSNAP-23 treated with PEP/rPKM2 matches to the peptide 92NFESGK97 of SNAP-23, suggesting that SNAP-23 Ser95 was phosphorylated.