Fig. 7

Fig. 7 JNK signaling pathway is activated in scamp5a KO zebrafish. A RNAseq analysis for identification of differentially expressed genes in scamp5a−/− larvae. Hierarchical clustering heatmap of changed genes in brains from 5 dpf WT and scamp5a−/− larvae. Dendrogram color code localizes selective cluster shown at higher magnification. WT: wild type larvae group, Mut: scamp5a−/− larvae group. B KEGG pathway analysis revealed that scamp5a KO affected expression of genes involved in neuroactive ligand-receptor interaction, calcium signaling pathway and MAPK signaling pathway. C The volcano plot of the MAPK signaling pathway genes in 5 dpf scamp5a−/− larvae brains compared to WT controls. False Discovery Rate (FDR) < 0.05. D mRNA extracts from 5 dpf larvae brains as indicated and were analyzed by RT-qPCR and the relative transcript levels of JNK-relative genes in scamp5a−/− larvae were compared to those in WT larvae. Housekeeping gene actb was used as internal control for RT-qPCR assay. n = 30 larvae per group for mRNA extracts. E, F WT, scamp5a−/− larvae were injected with plasmids as indicated at the one-cell stage. Brains extracts from 5 dpf larvae were harvested and analyzed by Western blot. E Lysates were probed with indicated antibodies. The primary antibody was anti-p-JNK, anti-t-JNK, anti-c-JUN. GAPDH was used as a loading control. n = 20 larvae per group for proteins extracts. Plasmid: GFP tag or human GFP-SCAMP5. F Relative expression levels of JNK-relative factors in indicated larvae were quantified by normalizing to those in WT larvae. G, H PC12 cells were transfected with siRNA as indicated for 72 h. Cell lysates were harvested and analyzed by Western blot. G Lysates were analyzed with the indicated antibodies. The primary antibody was anti-p-JNK, anti-t-JNK, anti-c-JUN. GAPDH was used as a loading control. H Relative expression levels of JNK-relative factors as indicated were quantified. Data in panel D, F and H are presented as mean ± SEM. n ≥ 3 independent experiments. Significance levels are denoted as follows: *P < 0.05, **P < 0.01, ***P < 0.001