Journal: Endocrinology

Article Title: AGEs-RAGE system down-regulates Sirt1 through the ubiquitin-proteasome pathway to promote FN and TGF-β1 expression in male rat glomerular mesangial cells.

doi: 10.1210/en.2014-1381

Figure Lengend Snippet: Figure 4. USP22 deubiquitinated Sirt1. A, The effects of AGEs (100 g/mL; 0, 3, 6, 12, and 24 hours) on USP22 protein expression. **, P .01; ***, P .001 vs 0 hour. B, GMCs were transfected with negative control, and 3 pairs of siRNA oligonucleotides targeting USP22 (75nM) for 48 hours, total protein was harvested and subjected to Western blot analysis. ***, P .001 vs control. C, The effects of USP22 depletion for 48 hours on Sirt1 expression. ***, P .001 vs control. D, The effects of USP22 depletion for 48 hours on Sirt1 ubiquitination. E, Immunofluorescence staining showed that USP22 was nuclear-localized protein in GMCs. Scale bars represented 20 m. F, GMCs were treated by AGEs (100 g/mL) for 12 hours to conduct Sirt1 coimmunoprecipitation reaction, the expression of Sirt1 and USP22 in Sirt1 immunoprecipitates and 10% WCL was detected by Western blot analysis. G, HEK-293T cells were cotransfected with Myc-Sirt1 and pRK5-HA-Ub, pRK5-HA-Ub K48, or pRK5-HA-Ub K63 for 48 hours. Then, Myc immunoprecipitation reaction was performed with anti-Myc antibody and immunoblotted with anti-HA antibody. H, USP22 deubiquitinated K48-linked ubiquitin chains on Sirt1 protein.

Article Snippet: PcDNA3-HA-Ub, pcDNA3-HA-Ub K0, vector pRK5, pRK5-HA-Ub, pRK5-HA-Ub K48, and pRK5-HA-Ub K63 were purchased from Addgene (http:// www.addgene.org/).

Techniques: Expressing, Transfection, Negative Control, Western Blot, Control, Ubiquitin Proteomics, Immunofluorescence, Staining, Immunoprecipitation

Journal: bioRxiv

Article Title: Requirement of FLCN tumor suppressor gene for mTORC1-mediated inhibition of TFE3 transcriptional activity

doi: 10.1101/2020.07.08.193169

Figure Lengend Snippet: (A) Wildtype (WT) and AMPK-null (AMPK-KO) MEFs were subjected to glucose deprivation for 30 min. Subcellular localization of TFE3 was determined by immunofluorescence staining. The graph shows the percentage of cells with nuclear staining of TFE3 and the statistical analysis. N>500, *P<0.001 (student’s t-test). (B) Fractionation experiment in HEK293 cells demonstrating the reduced level of cytoplasmic (Cyto) and increased level of nuclear (Nuc) TFE3 after glucose or amino acid deprivation for 1 hr. The graphs show the level of TFE3 in the cytoplasm and nucleus normalized to tubulin and topoisomerase 1 (Topo), respectively. N=3, #P<0.05, *P<0.01 (student’s t-test). WCL, whole cell lysate. (C) Hypo-phosphorylation and nuclear localization of TFE3 in response to glucose deprivation in NIH3T3, MDCK and HeLa cells. (D) Reduced phosphorylation levels of TFE3 and S6K1 in HEK293 cells treated with a glycolytic inhibitor, 2-deoxyglucose (2-DG, 20 mM).

Article Snippet: GST-p85-S6K1 expression vector was obtained from Addgene (Plasmid #8466) and transfected into HEK293A cells using Fugene 6 transfection reagent.

Techniques: Immunofluorescence, Staining, Fractionation

Journal: bioRxiv

Article Title: Requirement of FLCN tumor suppressor gene for mTORC1-mediated inhibition of TFE3 transcriptional activity

doi: 10.1101/2020.07.08.193169

Figure Lengend Snippet: (A) TFE3 phosphorylation by mTORC1 in vitro . 32 P-phosphorylation of GST-Tfe3 and GST-S6K1 but not GST by the immunoprecipitated mTORC1 kinase complex (HA-Raptor/myc-mTOR). PP242, 200 nM. IP, immunoprecipitates. (B) TFE3 phosphorylation by AMPK in vitro . GST-TFE3, SAMS peptide and GST were incubated with recombinant AMPK (α2β1γ1) in the presence or absence of AMP (400 μM). (C) Fractionation of HEK293 cells transfected with wildtype or mutant TFE3 expression vectors. The ratios of nuclear to cytoplasmic TFE3 (Nuc/Cyto) are indicated. (D) GST pull-down experiment demonstrating the interaction between wildtype TFE3 (but not TFE3-S321A mutant) and 14-3-3 protein. (E) FLCN expression was required for TFE3 phosphorylation in cells under nutrient-rich conditions. Both mTORC1 activity and FLCN expression were required for TFE3 phosphorylation. UOK257 cells expressing either FLCN or lacZ were treated with (F) DMSO (Cont), AICAR (2 mM), PP242 (1 μM) and (G) chloroquine (100 μM). (H) Schematic diagram demonstrating a proposed mechanism for TFE3 regulation by the FLCN-AMPK-mTORC1 signaling network.

Article Snippet: GST-p85-S6K1 expression vector was obtained from Addgene (Plasmid #8466) and transfected into HEK293A cells using Fugene 6 transfection reagent.

Techniques: In Vitro, Immunoprecipitation, Incubation, Recombinant, Fractionation, Transfection, Mutagenesis, Expressing, Activity Assay