pbs human irs1  (Addgene inc)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Family with sequence similarity 13, member A modulates adipocyte insulin signaling and preserves systemic metabolic homeostasis

doi: 10.1073/pnas.1720475115

Figure Lengend Snippet: Fam13a accelerates insulin signaling in adipocytes. (A) Immunoblotting for insulin signaling in 3T3-L1 adipocytes transfected with either negative control siRNA (Negative siRNA) or siRNA targeting Fam13a. Insulin-mediated Akt activation was quantified (n = 6 each). (B) Immunoblotting for insulin signaling in HEK293 cells transfected with either empty vector (MOCK) or the Fam13a expression construct (Fam13a-OE). Insulin-mediated Akt activation was quantified (n = 3 each). (C) Fam13a in 3T3-L1 adipocytes was immunoprecipitated, and coprecipitation of IRS1 was detected by immunoblotting. (D) FLAG-tagged Fam13a was immunoprecipitated in HEK293 cells transfected with the IRS1 expression construct and either empty vector (MOCK) or plasmid for Fam13a of whole-length or CCD-deletion mutants. Coprecipitation of IRS1 was detected by immunoblotting. (E) FLAG-tagged Fam13a was immunoprecipitated in HEK293 cells transfected with the IRS2 expression construct and empty vector (MOCK) or whole-length Fam13a. Coprecipitation of IRS2 was not detected. (F) HEK293 cells were transfected with either empty vector (MOCK) or the Fam13a expression construct (Fam13a-OE). Fam13a-overexpressing cells were cultured in the presence or absence of NT157. Cells were stimulated with insulin, and insulin signaling was analyzed by immunoblotting. Insulin-mediated Akt activation was quantified (n = 8 each). Data represent mean ± SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001, #not significant.

Article Snippet: The pBS human IRS1 (Addgene plasmid 11359) and pBABE puro mouse IRS2 myc (Addgene plasmid 11373) were gifts from Ronald Kahn, Joslin Diabetes Center, Boston. cDNAs for IRS1 and IRS2 were subcloned into pcDNA3 to prepare expression constructs.

Techniques: Western Blot, Transfection, Negative Control, Activation Assay, Plasmid Preparation, Expressing, Construct, Immunoprecipitation, Cell Culture

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Family with sequence similarity 13, member A modulates adipocyte insulin signaling and preserves systemic metabolic homeostasis

doi: 10.1073/pnas.1720475115

Figure Lengend Snippet: Fam13a enhances IRS1 expression by recruiting PP2A and inhibiting the proteasomal degradation. (A) Immunoblotting for IRS1 in HEK293 cells transfected with the IRS1 expression construct and either empty vector (MOCK) or the Fam13a expression construct (Fam13a-OE). IRS1 expression was quantified (n = 6 each). (B) Immunoblotting for IRS1 in 3T3-L1 adipocytes transfected with either negative control siRNA (Negative siRNA) or siRNA targeting Fam13a. IRS1 expression was quantified (n = 6 each). (C) Immunoblotting for IRS1 in HEK293 cells transfected with the IRS1 expression construct and either empty vector (MOCK) or the expression construct for Fam13a of whole-length or CCD1–2-deletion mutant. IRS1 expression was quantified (n = 6 each). (D) Immunoblotting for IRS1 in HEK293 cells transfected with the IRS1 expression construct and either empty vector (MOCK) or the Fam13a expression construct (Fam13a-OE). Cells were treated with vehicle or the proteasome inhibitor lactacystin. IRS1 expression was quantified (n = 4 each). (E) FLAG-tagged Fam13a was immunoprecipitated in HEK293 cells transfected with the IRS1 expression construct and empty vector (MOCK) or plasmid for Fam13a of whole-length or CCD-deletion mutants. Coprecipitation of PP2A B subunit was detected by immunoblotting. The same blot as the one used in Fig. 2D was used. (F) Immunoblotting for IRS1 in HEK293 cells transfected with the IRS1 expression construct and either empty vector (MOCK) or the Fam13a expression construct (Fam13a-OE). Cells were treated with vehicle or the PP2A inhibitor okadaic acid. IRS1 expression was quantified (n = 6 each). Data represent mean ± SEM. *P < 0.05, **P < 0.01, #not significant.

Article Snippet: The pBS human IRS1 (Addgene plasmid 11359) and pBABE puro mouse IRS2 myc (Addgene plasmid 11373) were gifts from Ronald Kahn, Joslin Diabetes Center, Boston. cDNAs for IRS1 and IRS2 were subcloned into pcDNA3 to prepare expression constructs.

Techniques: Expressing, Western Blot, Transfection, Construct, Plasmid Preparation, Negative Control, Mutagenesis, Immunoprecipitation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Family with sequence similarity 13, member A modulates adipocyte insulin signaling and preserves systemic metabolic homeostasis

doi: 10.1073/pnas.1720475115

Figure Lengend Snippet: Targeted deletion of Fam13a impairs metabolic health. (A) Body weight of male WT or Fam13a−/− mice fed NC at the indicated ages in weeks (n = 7 each). (B) Representative pictures for CT analysis were shown. (Scale bars, 10 mm.) (C) Body fat distribution in 17-wk-old male (n = 7 for WT; n = 5 for KO) and 20-wk-old female (n = 8 for WT; n = 9 for KO) mice. (D) ITT in 20-wk-old female WT and Fam13a−/− mice fed NC (n = 7 each). (E) IpGTT in 20-wk-old female WT and Fam13a−/− mice fed NC (n = 8 for WT; n = 9 for KO). (F) Quantitative real-time PCR for inflammatory genes in WAT of WT and Fam13a−/− mice fed NC (n = 8 each). (G) Immunoblotting for IRS1 in WAT of WT and Fam13a−/− mice fed NC (n = 7 for WT; n = 6 for KO). Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The pBS human IRS1 (Addgene plasmid 11359) and pBABE puro mouse IRS2 myc (Addgene plasmid 11373) were gifts from Ronald Kahn, Joslin Diabetes Center, Boston. cDNAs for IRS1 and IRS2 were subcloned into pcDNA3 to prepare expression constructs.

Techniques: Real-time Polymerase Chain Reaction, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Family with sequence similarity 13, member A modulates adipocyte insulin signaling and preserves systemic metabolic homeostasis

doi: 10.1073/pnas.1720475115

Figure Lengend Snippet: Loss of Fam13a exacerbates obesity-related metabolic disorder. (A) ITT in male WT and Fam13a−/− mice fed a HFD for 12 wk (n = 7 each). (B) IpGTT in male WT or Fam13a−/− mice fed a HFD for 12 wk (n = 6 for WT; n = 5 for KO). (C) Quantitative real-time PCR for inflammatory genes in WAT of WT and Fam13a−/− mice fed a HFD for 14 wk (n = 5 each). (D) Immunoblotting for insulin signaling in WAT of WT and Fam13a−/− mice fed a HFD for 12 wk. Insulin was s.c. administered, and WAT was isolated 20 min after insulin injection. Insulin-mediated Akt activation was quantified [n = 8 for WT-insulin (−); n = 11 for WT-insulin (+); n = 7 for KO-insulin (−); n = 12 for KO-insulin (+)]. (E) Immunoblotting for IRS1 in WAT of WT and Fam13a−/− mice fed a HFD for 12 wk. IRS1 expression was quantified (n = 6 each). (F) Adipocyte size in WAT of WT and Fam13a−/− mice fed a HFD for 14 wk (n = 4,500 adipocytes each). (G) Serum lipid profiles in WT and Fam13a−/− mice fed a HFD for 14 wk (n = 6 each). Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: The pBS human IRS1 (Addgene plasmid 11359) and pBABE puro mouse IRS2 myc (Addgene plasmid 11373) were gifts from Ronald Kahn, Joslin Diabetes Center, Boston. cDNAs for IRS1 and IRS2 were subcloned into pcDNA3 to prepare expression constructs.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Isolation, Injection, Activation Assay, Expressing