## 3 Results
### Relative Activity of BAY11-7082 and Ro106-9920 in NF-\(\kappa\)B Activation  
BAY11-7082 and Ro106-9920's effectiveness in reducing NF-\(\kappa\)B activation were further compared. As Figures 3 and 4 illustrate, BAY11-7082 (3 and 7 \(\upmu\)M) exhibited potent activity in inhibiting IKK, \(\mathrm{I}\mathrm{k}\mathrm{B}\alpha\), and p65 phosphorylation and reversing \(\mathrm{I}\mathrm{k}\mathrm{B}\alpha\) degradation for collagen-stimulated platelets. Nevertheless, Ro106-9920 (3 and 7 \(\upmu\)M) only affected p65 phosphorylation and \(\mathrm{I}\mathrm{k}\mathrm{B}\alpha\) degradation (Figure 4C,D) and had no effect on IKK and \(\mathrm{I}\mathrm{k}\mathrm{B}\alpha\)  
Figure 4: Effect of NF-kB activation by Ro106-9920 in platelets. Washed platelets (1.2 \(\times\) 10\({}^{9}\) cells/mL) were preincubated with Ro106-9920 (3 and 7 \(\upmu\)M) or a solvent control (0.1% DMSO), followed by collagen treatment (1 \(\upmu\)g/mL) to trigger (**A**) IKK, (**B**) IkBa, and (**C**) p65 phosphorylation or (**D**) IkBa degradation for immunoblotting study. Data are presented as mean \(\pm\) standard error of the mean (\(n\) = 4). **_p_ \(<\) 0.01, and ***_p_ \(<\) 0.001, compared with the resting control (group treated with Tyrode’s solution); **_p_ \(<\) 0.01, and ***_p_ \(<\) 0.001, compared with the 0.1% DMSO-treated group.  
Figure 3: The role of BAY11-7082 on NF-kB activation in platelets. Washed platelets (1.2 \(\times\) 10\({}^{9}\) cells/mL) were preincubated with BAY11-7082 (3 and 7 \(\upmu\)M) or a solvent control (0.1% DMSO), followed by collagen treatment (1 \(\upmu\)g/mL) to trigger (**A**) IKK, (**B**) IkBa, and (**C**) p65 phosphorylation or (**D**) IkBa degradation for immunoblotting study. Data are presented as mean \(\pm\) standard error of the mean (\(n\) = 4). *_p_ \(<\) 0.01, and ***_p_ \(<\) 0.001, compared with the resting control (group treated with Tyrode’s solution); *_p_ \(<\) 0.05, *_p_ \(<\) 0.01, and ***_p_ \(<\) 0.001, compared with the 0.1% DMSO-treated group.  
Effect of PTE, BAY11-7082, and Ro106-9920 in Immunostaining of p65 Phosphorylation by Confocal Microscopy  
As Figure 5 illustrates, the influence of PTE, BAY11-7082, and Ro106-9920 in NF-\(\kappa\)B activation was further confirmed by using confocal laser fluorescence microscopy to compare the direct immunostaining results of anti-p-p65 mAb (green fluorescence) with those of \(\alpha\)-tubulin (blue fluorescence) in resting or collagen-activated platelets. Collagen (1 \(\mu\)g/mL) increased p-p65 fluorescence intensity compared with the resting group. The intensity of p-p65 was significantly reduced in PTE-, BAY11-7082-, or Ro106-9920-treated groups compared with the resting control (Figure 5). However, \(\alpha\)-tubulin intensity did not alter among the groups (Figure 5).  
Figure 5: Activity of pterostilbene (PTE), BAY11-7082, and Ro106-9920 in regulating collagen-stimulated p65 phosphorylation in human platelets as determined through a confocal laser fluorescence study. Washed platelets (1.2 \(\times\) 10\({}^{9}\) cells/mL) were preincubated with PTE (7 \(\mu\)M), BAY11-7082 (7 \(\mu\)M), or Ro106-9920 (7 \(\mu\)M), followed by collagen treatment (1 \(\mu\)g/mL) to trigger platelet aggregation. The confocal images of p-p65 (green fluorescence) and \(\alpha\)-tubulin (blue fluorescence) were observed by using goat anti-rabbit CFTM 488A Dye and goat anti-mouse CFTM 405M Dye, respectively, as described in the Section 2. The profiles represent four similar studies. Bar: 7.5 \(\mu\)m.