Fig. 6From: Inhibition of lysophosphatidic acid receptor 1–3 deteriorates experimental autoimmune encephalomyelitis by inducing oxidative stressEffect of Ki16425 on proinflammatory cytokine milieu and oxidative stress in spinal cords after EAElow induction. A–M Protein lysates and total RNA were isolated from the lumbar spinal cords (n = 3 per group) of the sham, EAElow, EAElow + Ki16425 (15 and 30 mg/kg), and Ki16425 (30 mg/kg) groups at day 19–20 after immunization. A–C The lysates (n = 3 per group) were analyzed by Western blot using COX-2 (A), p-NF-κB (B), and p-p38 MAPK (C) antibodies to represent inflammatory signaling pathways and the results were quantified (A–C). D–F Total RNA (n = 3 per group) was analyzed by real-time PCR to measure mRNA expression of COX-2 (D), iNOS (E), and TNF-α (F) as representative cytokines. G–I Lysates of lumbar spinal cords (n = 3 per group) from sham, EAElow, EAElow + Ki16425 (15 and 30 mg/kg), and Ki16425 (30 mg/kg) groups at day 19–20 after immunization were analyzed by Western blot using 4-HNE antibody followed by quantification (G). Cryosections (n = 3 per spinal cord) of lumbar spinal cords (n = 3 per group) from each group were subjected to MitoSOX™ assay to measure mitochondrial superoxide level (H) followed by quantification (I). J–M Lumbar spinal cords (n = 3) from each group were analyzed by real-time PCR to investigate mRNA expression levels of NOX1 (J), NOX-2 (K), NOX3 (L), and NOX4 (M). Bar = 100 µm. Data are expressed as mean value, fold induction, or cell number ± SEM (ANOVA test; #p < 0.05 versus sham group; *p < 0.05 and **p < 0.01 versus EAElow group)Back to article page