Journal: Proceedings of the National Academy of Sciences of the United States of America
Article Title: MTSS1/Src family kinase dysregulation underlies multiple inherited ataxias
doi: 10.1073/pnas.1816177115
Figure Lengend Snippet: SFK dysregulation occurs in multiple SCAs. (A) Western blot of whole-cerebellum lysate from 15-wk-old mice shows a 95% reduction of in the band that corresponds to MTSS1 in ATXN1Q82 mice but only a 50% reduction in calbindin. Tubulin is included as a loading control. (B) Western blot of cerebellum lysate from 4-wk-old MIMEX15 mice shows no change in phospho-serine776 ATXN1 levels. (C) RNA-sequencing from ATXN1Q82 cerebella shows reduced fragments per kilobase of transcript per million mapped reads for Mtss1 mRNA in samples from 12-wk-old and 28-wk-old mice (*q < 0.005). (D) Mean firing frequency values (in Hertz) for WT and ATXN1Q82 mice, with and without dasatinib (Das) treatment. *P = 0.0094, one-way ANOVA with Tukey post hoc test. Error bars indicate SEM. (E) Western blot of whole-cerebellum lysate from 3-wk-old mice shows no change in MTSS1 in βIII-spectrin−/− mice, but active SFK-Y416 phosphorylation is increased. Calbindin and total Src are included as loading controls. (F) Mean firing frequency values (in Hertz) for wild-type and βIII-spectrin−/− mice, with and without dasatinib treatment. *P < 0.05, one-way ANOVA, Tukey post hoc test. Error bars indicate SEM. (G) SPTNB2 abundance is not changed in 4-wk-old MIMEX15 mice. (H) βIII-spectrin levels are reduced 40% in 24-wk-old ATXN2Q127 mice. (I) A model in which pathogenic alleles of ATNX1 (ATXN1Q82) and ATXN2 (ATXN2Q42) prevent the accumulation of MTSS1 and SPTBN2 which restrain SFK activity to prevent abnormal firing patterns and neurodegeneration.
Article Snippet: Rabbit anti-Src-Y416 (2101S or 6943S; Cell Signaling Technology), mouse anti–β-actin (Sigma), rabbit anti-Sptbn2 (PA1-46007; Thermo), rabbit anti-Atxn2 (HPA021146; Sigma), mouse anti-Atxn1 (ab63376; Abcam), rabbit anti-LC3A/B (4108; Cell Signaling Technology), rabbit anti-P62 (23214; Cell Signaling Technology), and rabbit anti-Src (2123 or 2108; Cell Signaling Technology) primary antibodies were detected with LICOR secondary antibodies.
Techniques: Western Blot, RNA Sequencing Assay, Activity Assay
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