Journal: PLoS Genetics

Article Title: Activin Signaling Targeted by Insulin/dFOXO Regulates Aging and Muscle Proteostasis in Drosophila

doi: 10.1371/journal.pgen.1003941

Figure Lengend Snippet: (A) Tissue-specific gene expression pattern of daw . (B) Tissue-specific distribution of transcription factor Smox using 7-day-old Oregon R females. (C–E) Lifespan analysis of Activin signaling using muscle-specific Gal4 driver (MHC-Gal4). Lifespan was extended by inactivating Activin genes ( daw , Smox and babo ) in muscle (Log-rank test, p <0.0001). (F–H) Lifespan analysis of Activin signaling using adult fat body-specific Gal4 driver (S106-GS-Gal4). Fat body-specific inactivation of Activin genes ( daw and Smox ) shortens lifespan (Log-rank test, p <0.0001). See for survival analysis. (I, J) mRNA expression of daw and phosphorylation of Smox are down-regulated by chico mutation and rescued by mutation of dFOXO. Muscle and fat body were dissected from 7-day-old female wildtype, chico −/− and chico;foxo double mutants. Band intensity was quantified using Bio-Rad Image Lab software. The average band intensity from four independent experiments is shown. Asterisk indicates significant difference between treatment and control ( p <0.05).

Article Snippet: Smox polyclonal antibody was generated against the peptide sequence (DSIVDYPLDNHTHQ) corresponding to amino acids 143–156 (Covance, Dedham, MA, USA) and affinity purified (Thermo/Pierce, Waltham, MA, USA) (specificity documented in ).

Techniques: Gene Expression, Expressing, Phospho-proteomics, Mutagenesis, Software, Control

Journal: PLoS Genetics

Article Title: Activin Signaling Targeted by Insulin/dFOXO Regulates Aging and Muscle Proteostasis in Drosophila

doi: 10.1371/journal.pgen.1003941

Figure Lengend Snippet: (A–C) Decline of flight with age is delayed in daw , Smox and babo RNAi flies. 40 females were scored for each genotype at each time point. Flying ability was measured at one week, three weeks and five weeks. (D) Poly-Ubiquitin-positive protein aggregates are reduced at old age in daw , Smox and babo RNAi flies. Aggregates were visualized with Poly-Ubiquitin FK2 antibody at one week, three weeks and five weeks. Scale bar: 20 µm. (E) RNAi for daw , Smox and babo preserves the decline of lysotracker-positive organelles (lysosomes). Scale bar: 20 µm. (F) Quantification of the cumulative area of protein aggregates for (n = 20). (G) Quantification of the number of lysotracker-positive stain for (n = 10). Asterisk indicates significant difference between treatment and control ( p <0.05).

Article Snippet: Smox polyclonal antibody was generated against the peptide sequence (DSIVDYPLDNHTHQ) corresponding to amino acids 143–156 (Covance, Dedham, MA, USA) and affinity purified (Thermo/Pierce, Waltham, MA, USA) (specificity documented in ).

Techniques: Ubiquitin Proteomics, Staining, Control

Journal: PLoS Genetics

Article Title: Activin Signaling Targeted by Insulin/dFOXO Regulates Aging and Muscle Proteostasis in Drosophila

doi: 10.1371/journal.pgen.1003941

Figure Lengend Snippet: (A) Autophagosomes indicated using an Atg8a -Cherry reporter in Activin RNAi flies or babo over-expressing ( babo-Act ) flies. 3-day old females. (B) Quantification of autophagosomes for (n = 20). (C) mRNA expression of autophagy genes ( Atg1 , Atg5 , Atg6 and Atg8a ) in aging muscle, at 10 days, 25 days and 45 days. (D) Phosphorylation of Smox in muscle increases with age. The average band intensity from three independent experiments was quantified using Image Lab software. (E) Inactivation of daw in muscle up-regulates autophagy gene expression. (F) RNAi against Smox in muscle up-regulates autophagy gene expression. (G) Constitutive activation of babo ( babo-Act ) in muscle inhibits autophagy gene expression. Asterisk indicates significant difference between treatment and control ( p <0.05).

Article Snippet: Smox polyclonal antibody was generated against the peptide sequence (DSIVDYPLDNHTHQ) corresponding to amino acids 143–156 (Covance, Dedham, MA, USA) and affinity purified (Thermo/Pierce, Waltham, MA, USA) (specificity documented in ).

Techniques: Expressing, Phospho-proteomics, Software, Gene Expression, Activation Assay, Control

Journal: PLoS Genetics

Article Title: Activin Signaling Targeted by Insulin/dFOXO Regulates Aging and Muscle Proteostasis in Drosophila

doi: 10.1371/journal.pgen.1003941

Figure Lengend Snippet: (A) Schematic of Atg8a genomic region (Smad box and ChIP-PCR target regions (P1–P3) are shown). Gray bar represents UTR and orange bar represents exon. (B) ChIP-PCR shows Smox binds to the promoter of Atg8a with binding enrichment calculated as the fold change of ChIP DNA vs. input DNA. The binding to the coding region of Actin gene (Act5C) was used as a negative control. (C) Smox binds to the promoter of Atg8a , but not Atg1 and Atg6 . The primers targeting the promoter regions containing putative Smad box in Atg8a , Atg1 and Atg6 were used in ChIP-PCR. (D) The binding of Smox to Atg8a promoter is abolished by chico mutation. Asterisk indicates significant difference between treatment and control ( p <0.05). (E). EMSA analysis reveals that recombinant Smox protein (MH1-DNA binding domain) binds to Smad binding element (AGAC AGAC) located in Atg8a promoter. Biotin-labeled Atg8a oligonucleotide probe ( 5′- CATATT AGAC AGAC ACCATT -3′ ) and its mutated forms are labeled with biotin. Halo-tagged Smox-MH1 DNA binding domain (amino acids 1–140) are expressed in E. coli and purified before used in EMSA analysis. (F) Recombinant Smox protein can also bind to mammalian SBE ( GTAT GTCT AGAC TGAA ).

Article Snippet: Smox polyclonal antibody was generated against the peptide sequence (DSIVDYPLDNHTHQ) corresponding to amino acids 143–156 (Covance, Dedham, MA, USA) and affinity purified (Thermo/Pierce, Waltham, MA, USA) (specificity documented in ).

Techniques: Binding Assay, Negative Control, Mutagenesis, Control, Recombinant, Labeling, Purification