Figure 4. Effects of B2 antagonist HOE 140 on phosphorylation sites of eNOS. Experiments were performed for 5 min in the absence and presence of HOE 140 (10^–6 M). In comparison with untreated odontoblasts, HOE 140 attenuated signal intensities for t-eNOS after 5 min in treated odontoblasts (control [A], 120.51 ± 8.41/HOE 140 [B], 104.21 ± 5.24). HOE 140 treatment induced a significant decrease in the signal intensities of p-eNOS at Ser¹¹⁷⁷ (control [C], 134.23 ± 2.28/HOE 140 [D], 94.29 ± 17.88). HOE 140 treatment decreased staining intensities of p-eNOS at Thr⁴⁹⁵ (control [E], 57.44 ± 10.77/HOE 140 [F], 28.31 ± 13.08). Incubations without primary antibodies resulted in an abolition of specific reaction products in odontoblasts of control (G) and HOE 140 treatment (H) conditions. The HOE 140 treatment did not significantly affect staining intensities of t-Akt/PKB in either control or treated odontoblasts (control [I], 143.14 ± 20.27/HOE 140 [J], 101.54 ± 28.80). The staining intensities of p-Akt/PKB in odontoblasts were significantly decreased after 5 min of HOE 140 treatment (control [K], 56.41 ± 9.30/HOE 140 [L], 28.74 ± 0.75). There were no significant differences in staining intensities for t-ERK1/2 (control [M], 131.64 ± 8.82/HOE 140 [N], 123.05 ± 11.37) and p-ERK1/2 (control [O], 27.65 ± 8.82/HOE 140 [P], 29.57 ± 9.38) between control and HOE 140-treated odontoblasts. Data are mean ± SD; n = 3 for each group. Significant differences were considered at a P value < 0.05. Bar = 70 µm.