## 3 Results
### Connection Between NF-\(\kappa\)B Signaling and MAPK Phosphorylation  
MAPK families play central roles in complex cellular programs, such as platelet activation, cell proliferation, differentiation and apoptosis [19]. At least three major MAPK families have been well characterized: ERK1/2, JNK1/2, and p38 MAPK. We employed various inhibitors for additional investigation on cellular signaling between NF-\(\kappa\)B and MAPKs. First, pretreatment with CBN (90 \(\mathrm{\SIUnitSymbolMicro m}\)M) reduced the phosphorylation of both ERK1/2 and JNK1/2, but not p38 MAPK, after stimulation with collagen (Figure 4A), which accords with previous findings [12]. Furthermore, both BAY11-7082 (8 \(\mathrm{\SIUnitSymbolMicro m}\)M) and CBN (90 \(\mathrm{\SIUnitSymbolMicro m}\)M) almost inhibited collagen-stimulated ERK1/2 and JNK1/2 phosphorylation (Figure 4B,C). Pretreatment with PD98059 (10 \(\mathrm{\SIUnitSymbolMicro m}\)M; ERK phosphorylation inhibitor) and SP600125 (10 \(\mathrm{\SIUnitSymbolMicro m}\)M; JNK phosphorylation inhibitor) reduced ERK1/2 and JNK1/2 phosphorylation, respectively (Figure 4B,C). Moreover, CBN (90 \(\mathrm{\SIUnitSymbolMicro m}\)M) and BAY11-7082 (8 \(\mathrm{\SIUnitSymbolMicro m}\)M) reduced collagen-stimulated \(\mathrm{I\kappa B\alpha}\) phosphorylation (Figure 5A,B). Interestingly, PD98059 (10 \(\mathrm{\SIUnitSymbolMicro m}\)M; Figure 5A) but not SP600125 (10 \(\mathrm{\SIUnitSymbolMicro m}\)M; Figure 5B) inhibited \(\mathrm{I\kappa B\alpha}\) phosphorylation, suggesting that NF-\(\kappa\)B and ERK1/2 activate each other, and JNK1/2 phosphorylation seems to be regulated by NF-\(\kappa\)B in collagen-activated platelets.  
Figure 2: Inhibitory activity of BAY11-7082, MG-132, and columbianadin in human platelet aggregation and ATP-release. Washed human platelets were preincubated with BAY11-7082 (IKK inhibitor; 4 and 8 \(\upmu\)M), MG-132 (proteasome inhibitor; 60 and 120 \(\upmu\)M), and CBN (45 and 90 \(\upmu\)M), followed by the addition of collagen (1 \(\upmu\)g/mL) to trigger (**A**) platelet aggregation and (**B**) ATP-release reaction (AU; arbitrary unit). The corresponding statistical data are illustrated in the low panel of figures. * \(p<0.05\), **\(p<0.01\) and *** \(p<0.001\), compared with the 0.1% DMSO-treated group (\(n=4\)).**  
Figure 3: Inhibition of NF-\(\kappa\)B activation by columbianadin in washed human platelets. (**A**) IxB\(\alpha\), (**B**) p65 phosphorylation, and (**C**) IxB\(\alpha\) protein degradation. * \(p<0.05\) and *** \(p<0.001\), compared with resting platelets; # \(p<0.05\) and *** \(p<0.001\), compared with the 0.1% DMSO-treated group (\(n=4\)).  
_Appl. Sci._**2020**, _10_, 7323  
Figure 4: Effects of BAY11-7082 on ERK1/2 and JNK1/2 phosphorylation in collagen-activated washed human platelets. Washed platelets were preincubated with 0.1% DMSO, CBN (90 \(\upmu\)M), BAY11-7082 (8 \(\upmu\)M), PD98059 (ERK inhibitor; 10 \(\upmu\)M) or SP600125 (JNK inhibitor; 10 \(\upmu\)M) and then treated with collagen (1 \(\upmu\)g/mL) to induce platelet activation. The levels of (**A**) ERK1/2, JNK1/2, and p38 MAPK phosphorylation were detected in the subcellular extracts of the collected platelets. The matching statistical data of (**B,C**) are displayed on the right panel of each figure. All phosphorylated proteins were normalized to the total proteins. * \(p<0.05\) and *** \(p<0.001\), compared with resting platelets; # \(p<0.05\), # \(p<0.01\), and *** \(p<0.001\), compared with the 0.1% DMSO-treated group (\(n=4\)).