Journal: Carcinogenesis

Article Title: ATM kinase activity modulates cFLIP protein levels: potential interplay between DNA damage signalling and TRAIL-induced apoptosis.

doi: 10.1093/carcin/bgq193

Figure Lengend Snippet: Fig. 3. p53 is not required for 5-FU ability to sensitize HCC cell lines to TRAIL-induced apoptosis. (A and C) HepG2-TR cells interfered (shp53) or not (shScramble) for p53 expression or incubated with pifithrin-a, a potent p53 inhibitor, were incubated in the absence on in the presence of 5-FU (A) or NCS (C) for the indicated times. cFLIP protein levels were revealed by immunoblotting with specific antibodies and quantified normalizing for each point the amount of cFLIP for the corresponding amount of tubulin. (B and D) Cells were then treated or not with 5-FU (B) or NCS (D) for 4 h and stimulated to undergo apoptosis with 10 ng/ml iz-TRAIL for 16 h. Apoptosis was determined as in Figure 1.

Article Snippet: The following antibodies and reagents were used: anti-phospho-Ser1981-ATM (Cell Signaling Technology, Beverly, MA), anti-ATM (2C1; Santa Cruz Biotechnology, Santa Cruz, CA), anti-FLIP (S and L) (H-202 rabbit polyclonal; Santa Cruz Biotechnology), anti-FLIP (S and L) (NF6; Alexis Biochemicals, Farmingdale, NY), anti-b-tubulin (Sigma, St Louis, MO), anti-p53 (Pab240; Santa Cruz Biotechnology), anti-ubiquitin (FK-2; Upstate, Billerica, MA). iz-TRAIL (human isoleucine zipper) has been kindly provided by H.Walczak (10), NCS, 5-FU and MG132 were commercially available (Sigma) as well as KU-55933 (Calbiochem, Merck, Darmstadt, Germany), whereas pifithrin-a was kindly provided by F.Maina.

Techniques: Expressing, Incubation, Western Blot

Journal: Carcinogenesis

Article Title: ATM kinase activity modulates cFLIP protein levels: potential interplay between DNA damage signalling and TRAIL-induced apoptosis.

doi: 10.1093/carcin/bgq193

Figure Lengend Snippet: Fig. 4. ATM activity selectively modulates cFLIPL protein stability through the proteasome pathway independently on p53. (A) ATM-deficient cells or ATM-proficient cells were incubated in the presence or in the absence of the proteasome inhibitor MG132 (10 lM) for 1 h. cFLIP protein levels were revealed by immunoblotting with specific antibodies. (B) ATM-proficient cells (C3ABR) were incubated with NCS in the presence or in the absence of MG132 and cFLIP protein levels analysed by immunoblotting. (C) C3ABR cells were pre-incubated with the ATM kinase inhibitor KU-55933 (10 lM, 16 h) and then treated with NCS for the indicated times. cFLIP proteins were revealed by immunoblotting. (D) C3ABR cells were pre-incubated in the presence or not of KU-55933 for 16 h, treated with NCS for 4 h and finally exposed to 500ng/ml iz-TRAIL. Apoptosis was determined by the analysis of DNA fragmentation upon propidium iodide nuclear staining 4 h after TRAIL treatment. (E) HepG2-TR cells interfered (shp53) or not (shScramble) for p53 expression were pre-incubated or not in the presence of MG132 or 30 min and then treated or not with NCS. cFLIP levels have been revealed by immunoblotting.

Article Snippet: The following antibodies and reagents were used: anti-phospho-Ser1981-ATM (Cell Signaling Technology, Beverly, MA), anti-ATM (2C1; Santa Cruz Biotechnology, Santa Cruz, CA), anti-FLIP (S and L) (H-202 rabbit polyclonal; Santa Cruz Biotechnology), anti-FLIP (S and L) (NF6; Alexis Biochemicals, Farmingdale, NY), anti-b-tubulin (Sigma, St Louis, MO), anti-p53 (Pab240; Santa Cruz Biotechnology), anti-ubiquitin (FK-2; Upstate, Billerica, MA). iz-TRAIL (human isoleucine zipper) has been kindly provided by H.Walczak (10), NCS, 5-FU and MG132 were commercially available (Sigma) as well as KU-55933 (Calbiochem, Merck, Darmstadt, Germany), whereas pifithrin-a was kindly provided by F.Maina.

Techniques: Activity Assay, Incubation, Western Blot, Staining, Expressing

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: 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: International journal of molecular medicine

Article Title: Xuebijing exerts protective effects on lung permeability leakage and lung injury by upregulating Toll-interacting protein expression in rats with sepsis.

doi: 10.3892/ijmm.2014.1943

Figure Lengend Snippet: Figure 1. Effect of XBJ on the mRNA expression of Tollip, IRAK1, TLR4, NF-κB65 and TRAF6 in lung tissue. Groups of mice were challenged with CLP and treated with XBJ 24 h later. The expression of Tollip, IRAK1, TLR4, NF-κB65 and TRAF6 in lung tissue was determined by RT-PCR. Representative RT-PCR shows the level of Tollip, IRAK1, TLR4, NF-κB65, and TRAF6 expression in the four rat groups. M, marker; A, normal control group; B, sham operation group; C, control group; D, treatment group.

Article Snippet: Rabbit anti-mouse Tollip, TLR4, TRAF6, p-IRAK1, VEGF-α, HO-1 and NF-κB polyclonal antibodies were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Marker, Control

Journal: International journal of molecular medicine

Article Title: Xuebijing exerts protective effects on lung permeability leakage and lung injury by upregulating Toll-interacting protein expression in rats with sepsis.

doi: 10.3892/ijmm.2014.1943

Figure Lengend Snippet: Figure 2. Administration of XBJ led to increased expression levels of Tollip mRNA, and inhibition of TLR4, NF-κB65 and TRAF6 mRNA expression in lung tissue in CLP-ALI mice. The expression of Tollip, IRAK1, TLR4, NF-κB65 and TRAF6 in lung tissue was determined by RT-PCR. Statistical summary of the densitometric analysis of Tollip, IRAK1, TLR4, NF-κB65 and TRAF6 expression in the four rat groups. Data are presented as mean ± stan dard deviation of one experiment consisting of three replicates. Experiments were performed in triplicate; ﹡P<0.05 and ﹡﹡P<0.01 vs. normal control group and sham operation group. ﹟P<0.05 and ﹟﹟P<0.01 vs. control group.

Article Snippet: Rabbit anti-mouse Tollip, TLR4, TRAF6, p-IRAK1, VEGF-α, HO-1 and NF-κB polyclonal antibodies were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Techniques: Expressing, Inhibition, Reverse Transcription Polymerase Chain Reaction, Control

Journal: International journal of molecular medicine

Article Title: Xuebijing exerts protective effects on lung permeability leakage and lung injury by upregulating Toll-interacting protein expression in rats with sepsis.

doi: 10.3892/ijmm.2014.1943

Figure Lengend Snippet: Figure 4. Administration of XBJ enhanced the expression of Tollip protein protein, and inhibition TLR4, NF-κB65, p-IRAK1 and TRAF6 protein expression in lung tissue in CLP-ALI mice. Groups of mice were challenged with LPS and treated with salidroside 24 h later. Tollip, p-IRAK1, TLR4, NF-κB65 and TRAF6 were assayed by western blot analysis. Statistical summary of the densitometric analysis of Tollip, p-IRAK1, TLR4, NF-κB65 and TRAF6 protein expression in the four rat groups. Data are presented as mean ± standard deviation of one experiment consisting of three replicates. Experiments were performed in triplicate; ﹡﹡P<0.01 vs. the normal control group and sham operation group. ﹟P<0.05, ﹟﹟P<0.01 vs. the control group.

Article Snippet: Rabbit anti-mouse Tollip, TLR4, TRAF6, p-IRAK1, VEGF-α, HO-1 and NF-κB polyclonal antibodies were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Techniques: Expressing, Inhibition, Western Blot, Standard Deviation, Control

Journal: International journal of molecular medicine

Article Title: Xuebijing exerts protective effects on lung permeability leakage and lung injury by upregulating Toll-interacting protein expression in rats with sepsis.

doi: 10.3892/ijmm.2014.1943

Figure Lengend Snippet: Figure 3. Administration of XBJ enhanced the expression of Tollip protein, and inhibition of TLR4, NF-κB65, p-IRAK1 and TRAF6 protein expression in lung tissue in CLP-ALI mice. Groups of mice were challenged with LPS and treated with salidroside 24 h later. Tollip, p-IRAK1, TLR4, NF-κB65 and TRAF6 were assayed by western blot analysis. Representative western blots show the level of Tollip, p-IRAK1, TLR4, NF-κB65 and TRAF6 protein expression in the four rat groups. A, normal control group; B, sham operation group; C, control group; D, treatment group.

Article Snippet: Rabbit anti-mouse Tollip, TLR4, TRAF6, p-IRAK1, VEGF-α, HO-1 and NF-κB polyclonal antibodies were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Techniques: Expressing, Inhibition, Western Blot, Control

Journal: Molecular Cell

Article Title: Initiation of Quality Control during Poly(A) Translation Requires Site-Specific Ribosome Ubiquitination

doi: 10.1016/j.molcel.2016.11.039

Figure Lengend Snippet:

Article Snippet: GST-UBE1 (human) , Boston Biochem , Cat. #E-306.

Techniques: Recombinant, Protease Inhibitor, Methylation, Ubiquitin Proteomics, Expressing, Plasmid Preparation, Sequencing, Negative Control, Software