Journal: Cell reports

Article Title: The insulin and IGF signaling pathway sustains breast cancer stem cells by IRS2/PI3K-mediated regulation of MYC

doi: 10.1016/j.celrep.2022.111759

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Article Snippet: Primary antibodies used in this study: rabbit anti-HA Tag (Cell Signaling Technology, Cat#3724; RRID: AB_1549585), rabbit anti-IRS1 (Bethyl Laboratories, Cat# A301-158A; RRID: AB_2125761), rabbit anti-IRS2 (Cell Signaling Technology, Cat# 4502; RRID: AB_2125774), rabbit anti-phospho-c-Myc (Thr58) (Cell Signaling Technology, Cat# 46650), rabbit anti-phospho-c-Myc (Ser62) (Cell Signaling Technology, Cat# 13748; RRID: AB_2687518), rabbit anti- GSK-3β (Cell Signaling Technology, Cat# 9315 RRID: AB_490890), rabbit anti-phospho-GSK-3β (Ser9) (Cell Signaling Technology, Cat# 9336; RRID: AB_331405), rabbit anti-insulin receptor β (Cell Signaling Technology, Cat# 3025; RRID: AB_2280448), rabbit anti-IGF-1 receptor β (Cell Signaling Technology, Cat# 3027, RRID: AB_2122378), rabbit anti-PI3 kinase p85 (Cell Signaling Technology, Cat# 4292; RRID: AB_329869), mouse anti-actin (Thermo Fisher Scientific, Cat# MA5-11869; RRID: AB_11004139), mouse anti-GAPDH (Santa Cruz Biotechnology, Cat# sc-32233; RRID: AB_627679), mouse anti-p-Tyr (Santa Cruz Biotechnology, Cat# sc-7020; RRID: AB_628123), mouse anti-α-tubulin (Sigma-Aldrich, Cat# T5168; RRID: AB_477579), mouse anti-Flag (Sigma-Aldrich, Cat# F3165; RRID: AB_259529).

Techniques: Control, Virus, Recombinant, Staining, cDNA Synthesis, Negative Control, CRISPR, Software

Journal: Nature Communications

Article Title: 6-Phosphogluconate dehydrogenase promotes mitochondrial fusion and immune suppression in tumor-associated monocytic suppressor cells

doi: 10.1038/s41467-025-68102-8

Figure Lengend Snippet: A MDSCs were generated in vitro using IL-6 (40 ng/mL) and GM-CSF (40 ng/mL) from bone marrow (BM) of Pgd fl/fl and Pgd fl/fl LysM Cre mice or wild type (WT) mice with 6AN (5 µM) or vehicle. Sorted MDSCs on day 4 were analyzed for 1318 signaling target proteins by Phospho-Explorer Antibody Array (Full moon BioSystem). B Oxidative PPP checkpoints were blocked by shRNA in WT BM-derived MDSCs. Phospho-IRS-1 S307 levels were examined on sorted M-MDSCs. Blots were processed parallel to samples from the same experiment. Results are representative of two repeats. C 6PG (10, 100, 1000 µM) was added to MDSC cell free lysates for 30 min and IRS-1 S307 phosphorylation measured by western blot. Blots were processed parallel to samples from the same experiment. Results are representative of two repeats. D , E Immunoprecipitated JNK1 and IRS-1 (substrate) were incubated with increasing 6PG concentrations (10, 100, 1000 µM) in an in vitro kinase assay ( D ). In the same experiment, JNK1-IRS-1 binding was evaluated by co-immunoprecipitation (Co-IP) using anti-JNK1 ( E ). Blots were processed parallel to samples from the same experiment. Results are representative of two repeats. F Serine to Alanine (S307A) site-specific mutation in IRS-1 was conducted in M-MDSCs (generated as in ( A )). Flowcytometry-sorted M-MDSCs were co-cultured with anti-CD3/anti-CD28 activated T cells (CFSE-labeled) at a 1:8 (M-MDSC: T cell) ratio. T cell proliferation was evaluated at 72 h. n = 3; One-way ANOVA. G , H M-MDSCs were generated as in ( A , F ). Mitochondrial ROS (mROS) production ( G ) and Arginase 1 expression ( H ) was examined on M-MDSC by flow cytometry. n = 3; One-way ANOVA. I Sorted M-MDSCs were prepared as in ( A , F ). Levels of PI3K (top), AKT (middle) and Drp1 S616 (bottom) phosphorylation were determined by western blot analysis. Blots were processed parallel to samples from the same experiment. Result is representative of two repeats. J – L MDSCs were generated as in ( A , F ), with N-Acetylcysteine (NAC: 0.5 mM) or vehicle (PBS). mROS production in M-MDSCs ( J ), suppressive capacity in co-culture with T cells ( K ) and Arginase expression levels ( L ) determined. n = 3; One-way ANOVA. All data are shown as mean ± SEM.

Article Snippet: The IRS-1 sequence containing a N-terminal HA tag was obtained by PCR using mouse IRS-1 cDNA as a template and cloned into the appropriate multiple cloning sites of the expression vector pcDNA 3.1(+) (CUSABIO).

Techniques: Generated, In Vitro, Ab Array, shRNA, Derivative Assay, Phospho-proteomics, Western Blot, Immunoprecipitation, Incubation, Kinase Assay, Binding Assay, Co-Immunoprecipitation Assay, Mutagenesis, Cell Culture, Labeling, Expressing, Flow Cytometry, Co-Culture Assay

Journal: Cell reports

Article Title: Runx2+ Niche Cells Maintain Incisor Mesenchymal Tissue Homeostasis through IGF Signaling

doi: 10.1016/j.celrep.2020.108007

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Article Snippet: Rabbit anti-Phospho-IRS-1 (Ser612) , Cell Signaling Technology , Cat# 3203; RRID:AB_1031167.

Techniques: Recombinant, Protease Inhibitor, TUNEL Assay, In Situ, Imaging, RNAscope, Multiplex Assay, cDNA Synthesis, Enzyme-linked Immunosorbent Assay, Software

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

Journal: Cancer cell

Article Title: Epigenetic silencing of CDR1as drives IGF2BP3-mediated melanoma invasion and metastasis

doi: 10.1016/j.ccell.2019.12.007

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit mAb IRS1 (D23G12) , Cell Signaling , Cat. # 3407; RRID:AB_2127860.

Techniques: Produced, Virus, Subcloning, Recombinant, Membrane, Western Blot, Autoradiography, RNA Extraction, Reverse Transcription, SYBR Green Assay, Sample Prep, Chromatin Immunoprecipitation, cDNA Synthesis, TA Cloning, Sequencing, RNAscope, Cell Culture, Control, DNA Methylation Assay, shRNA, Negative Control, Positive Control, Mutagenesis, Software