Fig 2: JWA regulates GLT-1 expression via ERK/MAPK and PI3K/Akt signaling pathways.a C8D1A cells were transfected with RFP-con or RFP-JWA. After 48 h, immunofluorescence for GLT-1 (green) was immunostained with anti-GLT-1 antibody. Scale bars = 200 µm. b C8D1A cells were transfected with si-con or si-JWA for various concentration (50 pmol or 100 pmol), followed by treatment with or without MPP+ 100 µM for 24 h. Expression levels of JWA and GLT-1 were detected by western blotting, GAPDH was used as a loading control to confirm that equal amounts of protein were loaded in each line (n = 3). c [3H]-Glutamate uptake assay was performed after C8D1A cells were transfected with si-JWA or Flag-JWA (n = 3). d C8D1A cells were transfected with si-JWA or Flag-JWA, followed by western blotting analysis to detect JWA, GLAST, GLT-1, phospho- and total-ERK, P38, JNK, and Akt proteins. The quantification for analysis of JWA, GLT-1, p-ERK, p-Akt protein (n = 3). Data are presented as mean ± SEM, one-way ANOVA, *P < 0.05, **P < 0.01

Fig 3: IU1 inhibits H/R-induced NF-?B and MAPK activation in trophoblast cells. (A) Western blotting was used to detect the protein levels of p-p65, NF-?B p65, p-p38, p38 MAPK, and USP14 in HTR8/Svneo cells treated with DMSO or 25 µM IU1 under H/R conditions. (B and C) The relative optical density of these genes was measured. **p < 0.01. DMSO, dimethyl sulfoxide; H/R, hypoxia/reoxygenation; NF-?B, nuclear factor kappa B

Fig 4: Ser blocks the IL-1ß-induced ASK1/P38/JNK activation in chondrocyte. Cells were exposed to L-1ß (10 ng/ml) with or without 25 µM Ser for 30 min. (A) Western blot and (B) quantitative analysis of ASK1/P38/JNK pathway in each group as above. ASK1, P38 and JNK were used as the external control. * p < 0.05, ** p < 0.01, n = 3.

Fig 5: Idebenone inhibited LPS-induced activation of MAPKs and NF-?B. (A) Expression levels of p-ERK, ERK, p-p38, P-38, p-JNK, and JNK were analyzed by western blotting. (B) Expression levels of p-NF-?B and NF-?B were analyzed by western blotting. (C) Idebenone inhibited LPS-caused translocation of p65 from cytosol to nucleus. BV2 microglial cells were immunostained with anti-NF-?B p65 antibodies and Alexa Fluor 594-conjugated secondary antibodies. Scale bar = 20 µm. Values are presented as the mean ± standard error. *p < 0.05, **p < 0.01, ***p < 0.001: idebenone + LPS vs. LPS; ##p < 0.01, ###p < 0.001: LPS vs. vehicle.