Overexpression of PAL31 in C6 effectively attenuated LPS/IFNγ stimulation in C6 or in C6 after co-culture with spinal neuron-glial cultures. (A) Level of nitrite release in C6 overexpressing GFP (C6/GFP) or GFP-tagged PAL31 (C6/PAL31) at 2 days after LPS/IFNγ stimulation (B) Levels of nitrite release in the transfected C6 co-cultured with spinal neuron-glial culture after LPS/IFNγ stimulation for 2 days. (C) Western blot analysis in C6/GFP or C6/PAL31 showing the expression of phosphorylated NF-kB is downregulated by PAL31 after LPS/IFNγ stimulation. NF-kB and GAPDH work as control. (D) Western blot analysis showing iNOS expression in the tranfected C6 co-cultured with spinal neuron-glial cultures after LPS/IFNγ stimulation. GAPDH works as a control. (E) Phosphorylated NF-kB-positive cells in LPS/IFNγ-stimulated neuron-C6 co-cultures (C6/GFP + neuron). (F) Phosphorylated NF-kB-positive cells in LPS/IFNγ-stimulated neuron-C6 co-cultures (C6/PAL31+ neuron). (G) Quantitative analysis of phosphorylated NF-kB positive cells in LPS/IFNγ-stimulated neuron-C6 co-culture shown in (E&F). C6 glial cultures were transfected with pGFP or pPAL31 before seeding spinal cord neurons. *P < 0.05, C6/GFP (+LPS/IFNγ) compared to C6/PAL31(+LPS/IFNγ), n = 3 ~ 4.