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: Oncotarget

Article Title: Sphingosine kinase 1 mediates AGEs-induced fibronectin upregulation in diabetic nephropathy

doi: 10.18632/oncotarget.20205

Figure Lengend Snippet: SphK1 was enriched by immunoprecipitation reaction in GMCs and subjected to western blot assay for detecting SphK1 ubiquitination (A) . Flag-SphK1 plasmid and pcDNA3-HA-Ub or pcDNA3-HA-Ub K0 plasmid was co-transfected in HEK-293A cells. After 3h of MG-132 treatment, cells were lysed to conduct immunoprecipitation reaction against SphK1 (B) . HEK-293A cells were co-transfected with Flag-SphK1 and vector pRK5, pRK5-HA-Ub, pRK5-HA-Ub K48, or pRK5-HA-Ub K63 for 48h. Then Flag immunoprecipitation reaction was performed with anti-Flag antibody and immunoblotted with anti-HA antibody (C) . CHX (1 μg/mL) was used to inhibit new protein synthesis in GMCs for detecting SphK1 stabilization (D) . ** P < 0.01, *** P < 0.001 vs. 0 h.

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: Immunoprecipitation, Western Blot, Ubiquitin Proteomics, Plasmid Preparation, Transfection

Journal: Journal of neurochemistry

Article Title: Degradation of gamma secretase activating protein by the ubiquitin-proteasome pathway

doi: 10.1111/jnc.13011

Figure Lengend Snippet: A. N2A-APPswe were cells transfected with empty vector or GSAP plasmid, then treated with the selective E3 UB ligase inhibitor SMER3 (15μM), the pan reversible inhibitor of deubiquitinases (DUBs) PR-619(10μM), and the proteasome inhibitors Z-IE (5μM) overnight. Upper panel. Cell lysates were immunoprecipitated (IP) with anti-GSAP antibody to pull down GSAP-associated proteins, the precipitates were then assayed by immunoblot analysis (IB) with anti-ubiquitin antibody to detect all the ubiquitinated GSAP. Lower panel. Total cell lysates from the condition described in A are directly assayed by Western blot (WB) with an antibody against ubiquitin. B. N2A-APPswe cells were transiently co-transfected with GSAP plus empty vector pRK5, or HA-tagged ubiquitin wilde type (WT), or HA-tagged ubiquitin mutant with only lysine 48 and mutation of all other lysines to arginines (K48), or HA-tagged ubiquitin mutant in which all lysines were mutated to arginines (KO). Upper panel. Cell lysates were first immunoprecipitated (IP) with anti-GSAP antibody to pull down GSAP-associated proteins, then the resultant immunoprecipated product was immunoblotted (IB) with an anti-HA antibody to detect all the ubiquitin in GSAP-containing immunoprecipitates. Lower panel. Total cell lysates from the condition described in B that are directly analyzed by Western Blot (WB) with anti-HA antibody and anti-GSAP antibody.

Article Snippet: For transfection, cells were grown to 70% confluence and separately transfected with 1 μg of empty vector pcDNA3.1 (Invitrogen, Carlsbad, CA), or GSAP cDNA (Origene, Rockville, MD), or empty vector pRK5 (Addgene, Cambridge MA), or pRK5-HA-Ubiquitin-WT (Addgene, Cambridge MA), or pRK5-HA-Ubiquitin-K48 (Addgene, Cambridge MA), or pRK5-HA-Ubiquitin-KO (Addgene, Cambridge MA), by using Lipofectamine® 2000 Transfection Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions and as previously described ( 5 , 7 ).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Mutagenesis