Figure 2.

SNAP-23 phosphorylation is not altered by IKK2 deletion and platelet activation is not reduced by pharmacological inhibition of IKK2. (A) Phosphorylation of S95 of SNAP-23 after stimulation of platelets with 100 µM PAR4-AP for 3 minutes and relative quantification of S95 of stimulated platelets (n = 5-6). (B) Overexpression of FLAG-tagged WT IKK2 or FLAG-tagged constructs mimicking exon 3 or exons 6 and 7 deletion in HeLa cells. Predicted band sizes for FLAG-tagged full-length (FL) IKK2: 90 kilodalton (kDa); for construct lacking exon 3 (Δ3): 7 kDa; without exons 6/7 (Δ6,7):17 kDa. (C) NF-κB reporter gene assay after transfection of the expression constructs. NanoLuc activity normalized to constitutively expressed β-galactosidase activity (n = 5-6). (D) Washed murine platelets were incubated with IKK inhibitors or respective solvent controls for 30 minutes and activated with the indicated concentrations of PAR4-AP. Median fluorescence intensity (MFI) of P-selectin (upper panel) and activated GPIIb/IIIa (lower panel) was analyzed (n = 3). (E) Washed murine platelets were incubated with the indicated concentrations of IKK inhibitors or respective dimethyl sulfoxide (DMSO) controls for 30 minutes and activated with 40 µM PAR4-AP. MFI of P-selectin (upper panels) and activated GPIIb/IIIa (lower panels) was analyzed (n = 6). (F) Human platelet-rich plasma was incubated with IKK inhibitors or respective DMSO control for 30 minutes and activated with 2.5 µM ADP, 5 µM thrombin receptor activator peptide 6 (TRAP-6), or 3 ng/mL CVX. MFI of P-selectin (left panel) and activated GPIIb/IIIa (right panel) was analyzed. n = 3. **P ≤ .01, ****P ≤ .0001.