Overexpression of PAL31 in mixed glial culture effectively reduced LPS/IFNγ stimulation after co-culture with spinal cord neuron-glial cultures. (A) ED-1- immunoreactive microglia/macrophage in confluent mixed glial cultures (B) S100- immunoreactive astroglia in confluent mixed glial cultures (C) LPS/IFNγ-stimulated neuron-glial co-cultures (Glia/GFP + neuron) showing expression of both iNOS and GFP (D) LPS/IFNγ-stimulated neuron-glial co-cultures (Glia/PAL31+ neuron) showing expression of both iNOS and PAL31. (E) Quantitative analysis of released nitric oxide, as a form of nitrite, in LPS/IFNγ-stimulated or non-stimulated neuron-glial co-culture. Mixed glial cultures were transfected with pGFP or pPAL31 before seeding spinal cord neurons. Co-cultures were then treated with LPS/IFNγ for 2 days. Medium were saved for nitrite level determination, while cells were processed for immunostaining with iNOS. *P < 0.05, n = 4. PAL31 overexpressing co-cultures (+LPS/IFNγ) compared to GFP overexpressing co-cultures (+LPS/IFNγ). Magnification 200X (A-D).