Journal: Cell Death & Disease
Article Title: Astrocytic JWA deletion exacerbates dopaminergic neurodegeneration by decreasing glutamate transporters in mice
doi: 10.1038/s41419-018-0381-8
Figure Lengend Snippet: a , b C8D1A cells were transfected with si-JWA or Flag-JWA. After 48 h, the mRNA levels of GLAST and GLT-1 were measured by quantitative real-time PCR. GAPDH was used as a control to normalize the differences in the amount of total RNA in each sample ( n = 3). c C8D1A cells were transfected with si-JWA or Flag-JWA, followed by western blotting analysis to detect transcription factor p-P65, YY1, phospho- and total-CREB proteins ( n = 3). d , e C8D1A cells were incubated with 50 µM of U0126 and/or 20 µM of LY for 3 h after transfect with Flag-con or Flag-JWA for 48 h, followed by western blotting analysis to detect p-ERK, p-Akt, p-CREB and GLT-1 ( n = 3). [ 3 H]-Glutamate uptake assay was also performed ( n = 3). f C8D1A cells were co-transfected with either Flag-con or Flag-JWA, together with the si-con or si-CREB. After 48 h, expression levels of 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). g SH-SY5Y cells were treated for 24 h with astrocyte CM from astrocytes transfected with si-con or from astrocytes transfected with si-JWA. After the treatment, SH-SY5Y cells were exposed to MPP + (50 μM or 100 μM) for 24 h. The expression levels of TH 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). Data are presented as mean ± SEM, one-way ANOVA, * P < 0.05, ** P < 0.01
Article Snippet: The mouse cerebellum astrocyte cell line C8D1A was purchased from the China Center for Type Culture Collection (Wuhan, China).
Techniques: Transfection, Real-time Polymerase Chain Reaction, Control, Western Blot, Incubation, Expressing
![a <t>C8D1A</t> 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 [ 3 H]-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](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4463/pmc05834463/pmc05834463__41419_2018_381_Fig5_HTML.jpg)
![a , b C8D1A cells were transfected with si-JWA or Flag-JWA. After 48 h, the mRNA levels of GLAST and GLT-1 were measured by quantitative real-time PCR. GAPDH was used as a control to normalize the differences in the amount of total RNA in each sample ( n = 3). c C8D1A cells were transfected with si-JWA or Flag-JWA, followed by western blotting analysis to detect transcription factor p-P65, YY1, phospho- and total-CREB proteins ( n = 3). d , e C8D1A cells were incubated with 50 µM of U0126 and/or 20 µM of LY for 3 h after transfect with Flag-con or Flag-JWA for 48 h, followed by western blotting analysis to detect p-ERK, p-Akt, p-CREB and GLT-1 ( n = 3). [ 3 H]-Glutamate uptake assay was also performed ( n = 3). f C8D1A cells were co-transfected with either Flag-con or Flag-JWA, together with the si-con or si-CREB. After 48 h, expression levels of 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). g SH-SY5Y cells were treated for 24 h with astrocyte CM from astrocytes transfected with si-con or from astrocytes transfected with si-JWA. After the treatment, SH-SY5Y cells were exposed to MPP + (50 μM or 100 μM) for 24 h. The expression levels of TH 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). Data are presented as mean ± SEM, one-way ANOVA, * P < 0.05, ** P < 0.01](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4463/pmc05834463/pmc05834463__41419_2018_381_Fig6_HTML.jpg)
