anti human ttp  (Santa Cruz Biotechnology)

Journal: BioMed Research International

Article Title: Ultrashort Wave Combined with Human Umbilical Cord Mesenchymal Stem Cell (HUC-MSC) Transplantation Inhibits NLRP3 Inflammasome and Improves Spinal Cord Injury via MK2/TTP Signalling Pathway

doi: 10.1155/2020/3021750

Figure Lengend Snippet: MK2 upregulation blocks the microglia polarisation promoted by combination therapy: (a) iNOS and arg-1 expression levels detected using Western Blotting; (b) contents of inflammatory factors (IL-6 and IL-10) determined using ELISA; (c) Western Blotting was performed on to determine the expression levels of TTP, NLRP3, pro-IL-1 β , and pro-IL-18; (d) contents of IL-1 β and IL-18 in cellular supernatant were quantified with ELISA. “ ∗ , #, &” indicated significant difference ( p < 0.05), “ ∗ ” vs. Sham group, “#” vs. SCI group, and “&” vs. rats treated with monotherapies and combination therapy.

Article Snippet: Membranes were then incubated with diluted primary antibodies against MK2 (ab131531, Abcam, UK), p-MK2 (ab131504, Abcam, UK), TTP (ab124024, Abcam, UK), NLRP3 (ab263899, Abcam, UK), pro-caspase-1 (ab179515, Abcam, UK), pro-IL-1 β (ab205924, Abcam, UK), iNOS (ab15323, Abcam, UK), arg-1 (ab272887, Abcam, UK), and GAPDH (ab8245, Abcam, UK) as loading internal control at 4°C for 12 h. On the next day, membranes were incubated with HRP-conjugated secondary antibody for 1 h at room temperature after rinse with TBST buffer solution for 3 changes.

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Molecular Genetics and Metabolism Reports

Article Title: Nuclear tristetraprolin acts as a corepressor of multiple steroid nuclear receptors in breast cancer cells

doi: 10.1016/j.ymgmr.2016.02.004

Figure Lengend Snippet: TTP overexpression represses ERα, PR, GR and AR transactivation in MCF-7 cells. Human breast cancer MCF-7 cells were transiently transfected with empty pCMV-3TAG vector (control) or with 0.25 μg of pCMV-3TAG-TTP (FLAG-TTP) along with the corresponding luciferase reporter vector. The effect of TTP on nuclear hormone receptors transactivation was determined by assay of luciferase activity, as described under “Experimental Procedures.” Assays were performed in triplicate in three independent experiments in the presence (white bars) or absence (black bars) of the corresponding nuclear receptor ligands (17β-estradiol for ERα, progesterone for PR, 5α-Androstan-17β-ol-3-on for AR and dexamethasone for GR. The results are represented as mean ± S.E. Differences in ERα activity between control MCF-7 cells and TTP-expressing MCF-7 cells are statistically significant ( p < 0.05).

Article Snippet: Human ERα antibody (Sc D -12, Sc HC-20) and Human TTP antibody (Sc-12565) were purchased from Santa Cruz Biotechnology, Inc. and TTP polyclonal (T5327) antibody was from Sigma-Aldrich.

Techniques: Over Expression, Transfection, Plasmid Preparation, Control, Luciferase, Activity Assay, Expressing

Journal: Molecular Genetics and Metabolism Reports

Article Title: Nuclear tristetraprolin acts as a corepressor of multiple steroid nuclear receptors in breast cancer cells

doi: 10.1016/j.ymgmr.2016.02.004

Figure Lengend Snippet: TTP interacts with ERα, PR, GR and AR in vivo . Endogenous ERα (A), PR (B), AR (C) and GR (C) were immunoprecipitated from protein extracts prepared from MCF-7 cells using specific antibodies. Immunoprecipitated proteins were resolved by PAGE, and the binding of TTP was visualized by Western blot. The input lane represents 10% of the protein extract used in the coimmunoprecipitation assays. IP: immunoprecipitation, WB: Western blot.

Article Snippet: Human ERα antibody (Sc D -12, Sc HC-20) and Human TTP antibody (Sc-12565) were purchased from Santa Cruz Biotechnology, Inc. and TTP polyclonal (T5327) antibody was from Sigma-Aldrich.

Techniques: In Vivo, Immunoprecipitation, Binding Assay, Western Blot

Journal: Molecular Genetics and Metabolism Reports

Article Title: Nuclear tristetraprolin acts as a corepressor of multiple steroid nuclear receptors in breast cancer cells

doi: 10.1016/j.ymgmr.2016.02.004

Figure Lengend Snippet: siRNA-mediated knockdown of TTP protein levels in MCF-7 cells. MCF-7 cells were transfected with specific siRNA-TTP (1.25 μg) or with an unrelated control siRNA (2.5 μg). (A) Protein extracts from MCF-7 cultures were resolved by PAGE, and expression levels of TTP, ERα, PR, GR, AR and tubulin, as a loading control protein, were evaluated by Western blot using specific antibodies as described under “Experimental Procedures.” TTP knockdown increases ERα, PR, GR and AR transactivation. MCF-7 cells were transfected with an unrelated siRNA (control) or a specific TTP-siRNA (1.5 μg), along with TK-LUC, in the presence of the corresponding nuclear receptor ligands. The effect of TTP-siRNA on ERα (B), PR (C), GR (D) and AR (E) transactivation was determined by a luciferase assay and compared with luciferase activity in MCF-7 cells transfected with empty pCMV-3TAG vector and the corresponding Tk-LUC reporter vector. Results, in triplicate in three independent experiments, are represented as mean ± S.E. (error bars). Differences in ERα activity in MCF-7 cells transfected with TTP or with TTP-siRNA were shown to be statistically significant ( p < 0.05).

Article Snippet: Human ERα antibody (Sc D -12, Sc HC-20) and Human TTP antibody (Sc-12565) were purchased from Santa Cruz Biotechnology, Inc. and TTP polyclonal (T5327) antibody was from Sigma-Aldrich.

Techniques: Knockdown, Transfection, Control, Expressing, Western Blot, Luciferase, Activity Assay, Plasmid Preparation

Journal: Breast Cancer Research and Treatment

Article Title: Tristetraprolin mediates the anti-proliferative effects of metformin in breast cancer cells

doi: 10.1007/s10549-016-3742-y

Figure Lengend Snippet: Metformin induces expression of TTP in both p53 wild-type and p53 mutant breast cancer cells. a , b Metformin increases TTP levels in p53 wild-type human breast cancer MCF7 cells. MCF7 cells were treated a with the indicated concentrations of metformin for 24 h or b with 6 mM metformin for the indicated length of time. The levels of TTP were measured by semi-qRT-PCR ( a , b , top ) and Western blotting ( a , b , bottom ). c Metformin induces TTP promoter activity in p53 wild-type MCF7 cells. MCF7 cells were transfected with pGL3/TTPp-1343 containing the TTP promoter. After treatment with 6 mM metformin for 24 h, luciferase activity was measured. The expression levels obtained from pGL3-transfected cells without metformin treatment were set to 1. Data are presented as the mean ± SD ( n = 3). *** p < 0.001. d Metformin induces p53 phosphorylation in p53 wild-type MCF7 cells but not p53 mutant MDA-MB-231 cells. MCF7 and MDA-MB-231 cells were treated with 6 mM metformin for the indicated length of time. The levels of p53 and phospho-p53 (p-p53) were measured by Western blotting. e , f Metformin increases TTP levels in p53 mutant human breast cancer MDA-MB-231 cells. MDA-MB-231 cells were treated e with the indicated concentrations of metformin for 24 h or f with 6 mM metformin for the indicated length of time. The levels of TTP were measured by semi-qRT-PCR ( e , f ; top ) and Western blotting ( e , f ; bottom ). g Metformin induces TTP promoter activity in MDA-MB-231 cells. MDA-MB-231 cells were transfected with pGL3/TTPp-1343 containing the TTP promoter. After treatment with 6 mM metformin for 24 h, luciferase activity was measured. The expression levels obtained from pGL3-transfected cells without metformin treatment were set to 1. Data are presented as the mean ± SD ( n = 3). *** p < 0.001

Article Snippet: Proteins were resolved by SDS–PAGE, transferred onto Hybond-P membranes (Amersham Biosciences Inc.), and probed with appropriate dilutions of the following antibodies: rabbit anti-human TTP (T5327, Sigma), anti-human c-Myc (sc-40, Santa Cruz), anti-p53 (1026-1, Epitomics), anti-phospho-p53 (#9284, Cell Signaling), anti-AMPK (#2603, Cell Signaling), anti-phospho-AMPK (#2535, Cell Signaling), anti-STAT3 (#12640, Cell Signaling), anti-phospho-STAT3 (#9134, Cell Signaling), and anti-β-actin (A2228, Sigma).

Techniques: Expressing, Mutagenesis, Quantitative RT-PCR, Western Blot, Activity Assay, Transfection, Luciferase

Journal: Breast Cancer Research and Treatment

Article Title: Tristetraprolin mediates the anti-proliferative effects of metformin in breast cancer cells

doi: 10.1007/s10549-016-3742-y

Figure Lengend Snippet: TTP mediates the anti-proliferative function of metformin in both p53 wild-type and p53 mutant breast cancer cells. a – d Metformin inhibits proliferation of both p53 wild-type MCF7 cells and p53 mutant MDA-MB-231 cells. a , c MCF7 and b , d MDA-MB-231 cells were treated ( a , b ) with the indicated concentrations of metformin for 24 h and ( c , d ) with 6 mM metformin for 24 and 48 h. Cell viability was assessed by measuring absorbance at 490 nm using an MTS cell proliferation assay. The values obtained with mock-treated cells were set to 100. Values are the mean ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001. e – h Inhibition of TTP attenuates the anti-proliferative effects of metformin in both MCF7 and MDA-MB-231 cells. MCF7 and MDA-MB-231 cells were transfected with e , g , h pcDNA6/TTP or f , g , h TTP- specific siRNA (TTP-siRNA). scRNA and pcDNA6 were used as negative controls. After treatment with 6 mM metformin for 24 h, cell viability was assessed by measuring the absorbance at 490 nm using an MTS cell proliferation assay. The values obtained with mock-treated cells were set to 100. Values are the mean ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001. ns not significant

Article Snippet: Proteins were resolved by SDS–PAGE, transferred onto Hybond-P membranes (Amersham Biosciences Inc.), and probed with appropriate dilutions of the following antibodies: rabbit anti-human TTP (T5327, Sigma), anti-human c-Myc (sc-40, Santa Cruz), anti-p53 (1026-1, Epitomics), anti-phospho-p53 (#9284, Cell Signaling), anti-AMPK (#2603, Cell Signaling), anti-phospho-AMPK (#2535, Cell Signaling), anti-STAT3 (#12640, Cell Signaling), anti-phospho-STAT3 (#9134, Cell Signaling), and anti-β-actin (A2228, Sigma).

Techniques: Mutagenesis, Proliferation Assay, Inhibition, Transfection

Journal: Breast Cancer Research and Treatment

Article Title: Tristetraprolin mediates the anti-proliferative effects of metformin in breast cancer cells

doi: 10.1007/s10549-016-3742-y

Figure Lengend Snippet: Metformin induces TTP expression through down-regulation of c - Myc . a Metformin treatment increases phospho-AMPK (pAMPK) but decreases c-Myc and phospho-STAT3 (pSTAT3) in MCF7 and MDA-MB-231 cells. MCF7 and MDA-MB-231 cells were treated with 6 mM metformin for the indicated length of time, and the levels of TTP, STAT3, pSTAT3, AMPK, pAMPK, and c-Myc were measured by Western blotting. b , c inhibition of c - Myc by siRNA enhances TTP promoter activity in MCF7 and MDA-MB-231 cells. b MCF7 and c MDA-MB-231 cells were transfected with pGL3/TTPp-1343 containing the TTP promoter. After treatment with 6 mM metformin for 24 h, luciferase activity was measured. The expression levels obtained from pGL3-transfected cells without metformin treatment were set to 1. Data are presented as the mean ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001. d Metformin increases TTP expression in an AMPK-dependent manner. MCF7 and MDA-MB-231 cells were treated with 6 mM metformin with or without 20 μM Compound C for 24 h. The levels of TTP, AMPK, and pAMPK were measured by Western blotting. e , f Overexpression of c-Myc blocks the effect of metformin on TTP induction. MDA-MB-231 cells were transfected with pcDNA3/c-Myc or control pcDNA3. Cells were treated with 6 mM metformin for 24 h. e c - Myc and f TTP levels were measured by quantitative RT-PCR. The values obtained with pcDNA-transfected and mock-treated cells were set to 1. Data are presented as the mean ± SD ( n = 3). *** p < 0.001. ns not significant. g , h Overexpression of c - Myc attenuates the anti-proliferative effect of metformin in MCF7 and MDA-MB-231 cells. g MCF7 and h MDA-MB-231 cells were transfected with pcDNA3/c-Myc or control pcDNA3. Cells were treated with 6 mM metformin for 24 h. Cell viability was assessed by measuring the absorbance at 490 nm using an MTS cell proliferation assay. The values obtained with mock-treated cells were set to 100. Data are presented as the mean ± SD ( n = 3). ** p < 0.01, *** p < 0.001. ns not significant

Article Snippet: Proteins were resolved by SDS–PAGE, transferred onto Hybond-P membranes (Amersham Biosciences Inc.), and probed with appropriate dilutions of the following antibodies: rabbit anti-human TTP (T5327, Sigma), anti-human c-Myc (sc-40, Santa Cruz), anti-p53 (1026-1, Epitomics), anti-phospho-p53 (#9284, Cell Signaling), anti-AMPK (#2603, Cell Signaling), anti-phospho-AMPK (#2535, Cell Signaling), anti-STAT3 (#12640, Cell Signaling), anti-phospho-STAT3 (#9134, Cell Signaling), and anti-β-actin (A2228, Sigma).

Techniques: Expressing, Western Blot, Inhibition, Activity Assay, Transfection, Luciferase, Over Expression, Quantitative RT-PCR, Proliferation Assay