Journal: Cell Death & Disease

Article Title: MDM2 facilitates adipocyte differentiation through CRTC-mediated activation of STAT3

doi: 10.1038/cddis.2016.188

Figure Lengend Snippet: CRTCs can bind STAT3, STAT5 and MDM2. ( a ) 293T cells were transfected with FLAG-tagged CRTCs and/or STATs. FLAG-immunoprecipitates (IPs) were analyzed for STAT3 and STAT5 by western blotting. ( b ) 293T cells were transfected with MYC-tagged MDM2 and/or STATs. MYC-IPs were analyzed for STAT3 and STAT5 by western blotting. ( c ) 293T cells were transfected with MYC-tagged MDM2 and/or JAK1. MYC-IPs were analyzed for JAK1 by western blotting. ( d ) 293T cells were transfected with FLAG-tagged CRTC2 and/or MDM2. FLAG-IPs were analyzed for MDM2 by western blotting. ( e ) 293T cells were transfected with FLAG-tagged CRTC3 and/or MDM2. FLAG-IPs were analyzed for MDM2 by western blotting

Article Snippet: pBABE-puro was a generous gift from Dr Ormond MacDougald. pBABE-puro C/EBPδ was described previously. pcDNA3 was kindly provided by Dr Jean-Christophe Marine. pCMV FLAG-MYC-CRTC2 was donated by Dr. Jeffrey Meier. pcDNA FLAG CRTC3 was a gift from Dr. Marc Montminy (Addgene, Cambridge, MA, USA; plasmid # 22976). pRK5 JAK1 was used previously. pcDNA3 STAT3 was a generous gift from Dr Jim Darnell (Addgene; plasmid #8706).

Techniques: Transfection, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: YAP transcriptionally regulates COX-2 expression and GCCSysm-4 (G-4), a dual YAP/COX-2 inhibitor, overcomes drug resistance in colorectal cancer

doi: 10.1186/s13046-017-0612-3

Figure Lengend Snippet: G-4 (10 μM) disturbed YAP-TEAD interaction. a G-4 treatment disturbed the YAP-TEAD1 interaction in the nucleus of HCT15/Tax cells. The YAP-TEAD1 interaction was probed in cells 4 h after G-4 treatment and in untreated cells using co-IP. b ChIP analysis of YAP interaction with the Cyr 61 and COX-2 promoter in HCT15/Tax cells. YAP was examined in cells 4 h after G-4 treatment and in untreated cells. ** P < 0.01 compared with vehicle group. c Cyr 61 and CTGF luciferase reporter activity was measured after treatment with G-4 for 48 h. The fold changes in luciferase activity were calculated by normalizing untreated cells with G-4-treated cells. Data are representative of at least three independent experiments. Error bars represent SD. ** P < 0.01 compared with control cells

Article Snippet: DNA plasmids that encode wild type human YAP (hYAP, CMV2-YAP) and TEAD1 vector (pRK5-Myc-TEAD 1) and YAP, COX-2, LATS1 shRNA were obtained from Addgene (USA).

Techniques: Co-Immunoprecipitation Assay, Luciferase, Activity Assay, Control

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