TLR4/MyD88/TRAF6 pathway was inhibited in hypoxic microglia with Notch signaling blockade.

<p><b>(A)</b> Notch blockade suppressed TLR4 mRNA expression. RT-PCR analysis showing the hypoxia induced increase in mRNA expression of TLR4 in primary microglia was significantly suppressed when pretreated with DAPT. (<b>B</b>) Western blotting of MyD88 and TRAF6 protein expression in BV-2 cells exposed to hypoxia for 2, 4, 6, 8, 12 and 24 h and control (c). The upper panel shows specific bands of MyD88 (38 k Da), TRAF6 (60 k Da) and β-actin (43 kDa). The lower panel is bar graphs showing significant changes in the optical density following hypoxic exposure. Note the MyD88 and TRAF6 protein expression after hypoxia is significantly increased. (<b>C</b>) RT-PCR analysis showing the hypoxia induced increase in mRNA expression of MyD88 and TRAF6 in primary microglia is significantly suppressed when pretreated with DAPT. (<b>D</b>) Western blotting of MyD88 and TRAF6 protein expression in BV-2 cells exposed to hypoxia for 8 h, hypoxic BV-2 cells pretreated with DAPT and corresponding control (c). The bar graphs showing increase in MyD88 and TRAF6 protein expression induced by hypoxia is significantly suppressed in DAPT pretreated group. Significant difference between control vs hypoxia groups is shown as *<i>p</i><0.05 and **<i>p</i><0.01; significant difference between hypoxia vs hypoxia+DAPT groups is shown as ^#<i>p</i><0.05 and ^##<i>p</i><0.01. The values represent the mean ±SD in triplicate.