Journal: PLoS ONE

Article Title: Trichostatin A Modulates Thiazolidinedione-Mediated Suppression of Tumor Necrosis Factor α-Induced Lipolysis in 3T3-L1 Adipocytes

doi: 10.1371/journal.pone.0071517

Figure Lengend Snippet: 3T3-L1 adipocytes were treated with vehicle (Control), 1 µM Rosi (Rosi), 10 ng/ml TNFα (TNFα), or both (Rosi+TNFα), together with vehicle (DMSO, bars 1-4) or 660 nM TSA (TSA, bars 5-8) for 24 h. Glycerol released into the media and protein concentrations of cell lysate were determined as described in Materials and Methods . Each point represents the mean ± S.E. of seven independent experiments. Asterisks denote significant differences (***p<0.001). NS, not significant. ## p<0.01 bar 5 vs 1.

Article Snippet: Recombinant murine TNFα (No. 410-MT) was from R & D Systems (Minneapolis, MN).

Techniques: Control

Journal: PLoS ONE

Article Title: Trichostatin A Modulates Thiazolidinedione-Mediated Suppression of Tumor Necrosis Factor α-Induced Lipolysis in 3T3-L1 Adipocytes

doi: 10.1371/journal.pone.0071517

Figure Lengend Snippet: 3T3-L1 adipocytes were treated with vehicle (Control), 1 µM Rosi (Rosi), 10 ng/ml TNFα (TNFα), or both (Rosi+TNFα), together with increasing TSA doses (0, 6.6, 66, 660, 6600 nM) for 24 h. Glycerol released into the media and protein concentrations of cell lysate were determined as described in Materials and Methods . Each point represents the mean ± S.E. of three independent experiments. Asterisks denote significant differences (p<0.05). NS, not significant. # p<0.05 vs 0 nM control.

Article Snippet: Recombinant murine TNFα (No. 410-MT) was from R & D Systems (Minneapolis, MN).

Techniques: Control

Journal: PLoS ONE

Article Title: Trichostatin A Modulates Thiazolidinedione-Mediated Suppression of Tumor Necrosis Factor α-Induced Lipolysis in 3T3-L1 Adipocytes

doi: 10.1371/journal.pone.0071517

Figure Lengend Snippet: (A) 3T3-L1 adipocytes were transfected with non-targeting luciferase siRNA (Luc), or siRNA against SMRT, NCoR, or PPARγ. The levels of mRNA were determined by qPCR. Each point represents the mean ± S.E. of at least three independent experiments. (B) 3T3-L1 adipocytes were transfected with control (Luc), SMRT, NCoR, or PPARγ siRNA. 24 h post transfection, cells were treated with vehicle (Control), 1 µM Rosi (Rosi), 10 ng/ml TNFα (TNFα), or both (Rosi+TNFα) for additional 24 h. Glycerol released into the media and protein concentrations of cell lysate were determined as described in Materials and Methods . Each point represents the mean ± S.E. of four independent experiments. Asterisks denote significant differences (*p<0.05; **p<0.01). NS, not significant.

Article Snippet: Recombinant murine TNFα (No. 410-MT) was from R & D Systems (Minneapolis, MN).

Techniques: Transfection, Luciferase, Control

Journal: PLoS ONE

Article Title: Trichostatin A Modulates Thiazolidinedione-Mediated Suppression of Tumor Necrosis Factor α-Induced Lipolysis in 3T3-L1 Adipocytes

doi: 10.1371/journal.pone.0071517

Figure Lengend Snippet: (A) 3T3-L1 adipocytes were treated with DMSO, 660 nM TSA, 20 µM SAHA, 10 µM MS275, or 5 µM MC1568 for 24 h. Cellular proteins were solubilized and subjected to SDS-PAGE and Western blot analysis with the indicated antibodies. Samples were treated in duplicate. Representative immunoblots from three independent experiments are shown. (B) 3T3-L1 adipocytes were treated with vehicle (Control), 1 µM Rosi (Rosi), 10 ng/ml TNFα (TNFα), or both (Rosi+TNFα), together with vehicle (DMSO), 660 nM TSA, 10 µM MS275, 5 µM MC1568, or combination of MS275 and MC1568 (MS275+MC1568) for 24 h. (C) 3T3-L1 adipocytes were treated with vehicle (Control), 1 µM Rosi (Rosi), 10 ng/ml TNFα (TNFα), or both (Rosi+TNFα), together with vehicle (DMSO), 660 nM TSA (TSA), 5 or 20 µM SAHA (SAHA) for 24 h. Glycerol released into the media and protein concentrations of cell lysate were determined as described in Materials and Methods . Each point represents the mean ± S.E. of four independent experiments. Asterisks denote significant differences (*p<0.05; **p<0.01; ***p<0.001). NS: not significant. # p<0.05, ## p<0.01 vs the corresponding DMSO Control.

Article Snippet: Recombinant murine TNFα (No. 410-MT) was from R & D Systems (Minneapolis, MN).

Techniques: SDS Page, Western Blot, Control

Journal: PLoS ONE

Article Title: Trichostatin A Modulates Thiazolidinedione-Mediated Suppression of Tumor Necrosis Factor α-Induced Lipolysis in 3T3-L1 Adipocytes

doi: 10.1371/journal.pone.0071517

Figure Lengend Snippet: (A and B) 3T3-L1 adipocytes were pretreated with vehicle (DMSO) or 660 nM TSA, together with or without 1 µM Rosi (Rosi) for 24h. Cells were then treated with or without 10 ng/ml TNFα for 30 min. Cellular proteins were solubilized and subjected to SDS-PAGE and Western analysis with the indicated antibodies. Representative immunoblots and quantification data from five independent experiments are shown in 5A and B, respectively. (C) ERK phosphorylation correlates highly with lipolysis in the treatments of Rosi, TNFα, or both in the presence or absence of TSA, as shown by fitting with linear regression. Individual values were obtained from the experiments described in Figures. 1 and 7B.

Article Snippet: Recombinant murine TNFα (No. 410-MT) was from R & D Systems (Minneapolis, MN).

Techniques: SDS Page, Western Blot, Phospho-proteomics

Journal: PLoS ONE

Article Title: Trichostatin A Modulates Thiazolidinedione-Mediated Suppression of Tumor Necrosis Factor α-Induced Lipolysis in 3T3-L1 Adipocytes

doi: 10.1371/journal.pone.0071517

Figure Lengend Snippet: (A) 3T3-L1 adipocytes were pretreated with vehicle (DMSO) or 25 µM U0126 for 1 h, followed by treatment with or without 10 ng/ml TNFα for 30 min. Cellular proteins were solubilized and subjected to SDS-PAGE and Western blot analysis with the indicated antibodies. (B) 3T3-L1 adipocytes were treated with vehicle (Control), 1 µM Rosi (Rosi), 10 ng/ml TNFα (TNFα), or both (Rosi+TNFα), together with DMSO (bars 1-4), 660 nM TSA (bars 5-8), 25 µM U0126 (bars 9-12), or both (bars 13-16) for 24h. Glycerol released into the media and protein concentrations of cell lysate were determined as described in Materials and Methods . Each point represents the mean ± S.E. of four independent experiments. Asterisks denote significant differences (**: p<0.01). NS, not significant. ## p<0.01 vs Treatment No.1.

Article Snippet: Recombinant murine TNFα (No. 410-MT) was from R & D Systems (Minneapolis, MN).

Techniques: SDS Page, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: After Ischemic Stroke, Minocycline Promotes a Protective Response in Neurons via the RNA-Binding Protein HuR, with a Positive Impact on Motor Performance

doi: 10.3390/ijms24119446

Figure Lengend Snippet: Table of the antibodies used in the experiments.

Article Snippet: Anti-TNFα , TNFα , rabbit monoclonal , Abcam , ab205587 , for WB we uded 1:500 , primary antibodies.

Techniques: Marker

Journal: Nanomaterials

Article Title: Few Layer Graphene Does Not Affect Cellular Homeostasis of Mouse Macrophages

doi: 10.3390/nano10020228

Figure Lengend Snippet: Cytokine secretion by BMDMs. ( A ) IL-6, ( B ) TNF-α, and ( C ) IL-1β levels in cell supernatant after treatment with or without FLG (3, 10, 30, 100 µg/mL), and LPS (1 µg/mL) for 24 h. A One-way ANOVA followed by Bonferroni’s post-test was performed to determine the statistical differences for control untreated cells versus FLG-treated samples and LPS-treated cells (* p < 0.05).

Article Snippet: In short, polyvinyl microtiter 96-well plates (Falcon) were coated overnight at 4 °C with 50 µL/well of purified rat anti-mouse IL-6 (0.5 mg/mL, BD Pharmingen #554400), rabbit anti-mouse TNF-α (0.5 mg/mL, BD Pharmingen #557516) and IL-1β (1 mg/mL, BD Biosciences, Le Pont de Claix, France), #51_26661E), diluted in coating buffer (Carbonate/bicarbonate buffer 0.05 M, pH 9.6).

Techniques:

Journal: BioMed Research International

Article Title: Imbalance of the Nerve Growth Factor and Its Precursor as a Potential Biomarker for Diabetic Retinopathy

doi: 10.1155/2015/571456

Figure Lengend Snippet: Shedding of p75 NTR is consistent in vitreous and serum. (a) Representative bands show p75 NTR expression in vitreous and serum for diabetic compared to nondiabetic control groups. The full length p75 NTR (75 kD) and receptor ectodomain (50 kD) had similar levels of expression in control and diabetic (DB) groups of both vitreous and serum. The possible proteolytic C terminal fragment (CTF) and intracellular domain (ICD) appeared at 27 kD and 22 kD. Differences in expression patterns between vitreous and serum as well as between diabetic and control groups were evident for both CTF and ICD. (b) In vitreous, 27 kD p75 NTR receptor fragment was significantly increased in diabetic (1.65-fold ± 0.23) compared to nondiabetic control group ( N = 4–11, * P < 0.05). (c) In serum, a significant increase in 22 kD p75 NTR occurred in diabetic samples (1.85-fold ± 0.30) compared to nondiabetic controls ( N = 6–10, * P < 0.05).

Article Snippet: The following antibodies were used for immunoblotting: rabbit polyclonal anti-NGF and anti-proNGF (Alomone Labs, Israel) and rabbit polyclonal p75 NTR (1 : 5000), a kind gift from Dr. Bruce Carter, Vanderbilt University, Nashville, TN.

Techniques: Expressing

Journal: BioMed Research International

Article Title: Imbalance of the Nerve Growth Factor and Its Precursor as a Potential Biomarker for Diabetic Retinopathy

doi: 10.1155/2015/571456

Figure Lengend Snippet: Expression of p75 NTR receptor is consistent in vitreous and serum. Results are shown for p75 NTR expression in vitreous and serum of diabetic (DB) and control participants normalized to Ponceau S and respective controls. Full length p75 NTR receptor (75 kD) was not significantly different in diabetic sample compared to control groups in either (a) vitreous ( N = 4–11) or (b) serum ( N = 6–9). The p75 NTR ectodomain (50 kD) was also not significantly different in diabetic compared to control groups in (c) vitreous ( N = 4–11) or (d) serum ( N = 6–9).

Article Snippet: The following antibodies were used for immunoblotting: rabbit polyclonal anti-NGF and anti-proNGF (Alomone Labs, Israel) and rabbit polyclonal p75 NTR (1 : 5000), a kind gift from Dr. Bruce Carter, Vanderbilt University, Nashville, TN.

Techniques: Expressing

Journal: Purinergic Signalling

Article Title: Up-regulation of P2X7 receptors mediating proliferation of Schwann cells after sciatic nerve injury

doi: 10.1007/s11302-015-9445-8

Figure Lengend Snippet: Expression of P2X7R ir in longitudinal sections of normal sciatic nerves. a–c show co-localization of P2X7R ir (red) and S100Aβ ir (green). Note an arrowhead showing a fibre-like structure and an arrow showing a trapezoid structure in a and b; c is the merged image of a and b. Note an arrow indicating a trapezoid structure double labelled by both P2X7R and S100β antibodies (yellow). d–f show co-localization of P2X7R ir (red) and Tuj-1 ir (green). Note an arrow showing a trapezoid structure in d and an arrow showing an axon in e. f is the merged image of d and f; note an arrow indicating a green axon passing through the middle of five trapezoid structures. g–i show co-localization of P2X7R ir (red) and p75NTR ir (green). Note an arrow showing a fibre-like structure in g and h. i is the merged image of g and h. An arrow indicates a double-labelled non-myelinating Schwann cell with P2X7R and p75NTR antibodies (yellow). j–l show co-localization of P2X7R ir (red) and MBP ir (green). Note an arrow showing a fibre-like structure with P2X7R ir, which was not labelled by MBP in l. m–o show co-localization of P2X7R ir (red) and CASPR ir (green). Note that no colocalization of P2X7R ir and CASPR ir was observed. Scale bars in a–c and g–i = 100 μm; scale bars in d–f = 50 μm

Article Snippet: The sections were washed 3–5 min in PBS, and then preincubated in a blocking solution (10 % normal bovine serum, 0.2 % Triton X-100, 0.4 % sodium azide in 0.01 mol/L PBS, pH 7.2) for 30 min followed by incubation with the primary antibodies: P2X7R (1:1000), Alomone, rabbit polyclonal, APR-004; S100β (1:400), Tuj-1 (1:200), p75NTR, PCNA (1:200), MBP (1:200), mouse monoclonal antibodies from Boster, CASPR (1:400) mouse monoclonal antibodies from Santa Cruz, at room temperature overnight.

Techniques: Expressing

Journal:

Article Title: Anti-inflammatory effects of a new tumour necrosis factor-alpha (TNF-?) inhibitor (CNI-1493) in collagen-induced arthritis (CIA) in rats

doi: 10.1046/j.1365-2249.1999.00750.x

Figure Lengend Snippet: Immunostaining of synovial tissue in CNI-1493-treated and non-treated DA rats with CIA. Immunostaining of TNF-α (A,C) and MHC II (B,D) in knee joint tissue from rats with CIA. Synovitis, articular cartilage and bone (dark blue staining) are evident in all figures. (A,B) Stainings in consecutive sections from an animal treated daily with CNI-1493 from start of immunization (rat V in Table 3). (C,D) Consecutive sections in tissue from a placebo-treated animal (rat I in Table 3). Both animals were killed on day 21 post-immunization (p.i.), when maximal signs of inflammation occurred in the placebo-treated group of animals. A massive infiltration of MHC II+ macrophages was recorded in the synovitis of both the CNI-treated (B) and the placebo-treated (D) rat, Yet TNF-α-expressing cells were only abundant in the placebo-treated animal (C), while the number of these cells was dramatically reduced in the CNI-treated rat (A).

Article Snippet: After additional thorough washes in BSS–saponin, sections were incubated overnight at room temperature in a humidified chamber with 50 μl of cytokine-specific antigen affinity-purified antibody (either polyclonal rabbit anti-rat TNF-α (lot no. 8-14; Dr P. van der Meide, Biomedical Primate Research Centre, Rijswijk, The Netherlands), or polyclonal antigen affinity-purified goat anti-rat TNF-α (AF-510; R&D Systems, Minneapolis, MN; used at 2 μg/ml).

Techniques: Immunostaining, Staining, Expressing

Journal: Experimental and Therapeutic Medicine

Article Title: Inhibition of the NMDA receptor protects the rat sciatic nerve against ischemia/reperfusion injury

doi: 10.3892/etm.2016.3148

Figure Lengend Snippet: Effects of MK-801 on tumor necrosis factor (TNF)-α protein expression levels in the rat sciatic nerve (SN) following ischemia/reperfusion (I/R) injury. Protein expression levels of TNF-α were determined using immunohistochemistry (magnification, ×400). (A) No TNF-α expression was detected in the Schwann cells derived from the SN of a sham-operated rat. (B) Moderate protein expression levels of TNF-α were detected in the Schwann cells derived from the SN fiber of a rat in the 12 h post-reperfusion I/R subgroup. (C) Higher protein expression levels of TNF-α were detected in the Schwann cells derived from the SN of a rat in the 24 h post-reperfusion I/R subgroup. (D) Numerous inflammatory cells had infiltrated the area surrounding the Schwann cells and moderate protein expression levels of TNF-α were detected in the SN of a rat in the 72 h post-reperfusion I/R subgroup. (E) Widespread demyelination and mild-to-moderate TNF-α protein expression levels in Schwann cells were detected in the SN derived from a rat in the 7 days post-reperfusion I/R subgroup. (F) A SN from a rat in the I/R + MK-801 group at 12 h post-reperfusion exhibited mild-to-moderate TNF-α protein expression levels in Schwann cells. (G) A SN from a rat in the I/R + MK-801 group at 24 h post-reperfusion exhibited markedly fewer infiltrating cells, as compared with the SN derived from I/R rats at the same time point post-reperfusion. Moderate protein expression levels of TNF-α expression were observed. (H) A SN derived from a rat in the I/R + MK-801 group at 7 days post-reperfusion. As compared with the SNs derived from the I/R rats, the extent of demyelination was markedly reduced and Schwann cells exhibited only low protein expression levels of TNF-α.

Article Snippet: Tissue slices were incubated with rabbit anti-mouse TNF-α polyclonal antibody (1:100; BA14901; Wuhan Boster Bio-Engineering Co., Ltd.) at 4°C overnight.

Techniques: Expressing, Immunohistochemistry, Derivative Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Inhibition of the NMDA receptor protects the rat sciatic nerve against ischemia/reperfusion injury

doi: 10.3892/etm.2016.3148

Figure Lengend Snippet: Protein expression levels of tumor necrosis factor-α in the various treatment subgroups were quantified using the integrated optical density method, and are presented as the mean ± standard deviation (n=6). Δ P<0.05, ΔΔ P<0.01 vs. the I/R group. I/R, ischemia reperfusion.

Article Snippet: Tissue slices were incubated with rabbit anti-mouse TNF-α polyclonal antibody (1:100; BA14901; Wuhan Boster Bio-Engineering Co., Ltd.) at 4°C overnight.

Techniques: Expressing, Standard Deviation

Journal: Experimental and Therapeutic Medicine

Article Title: Inhibition of the NMDA receptor protects the rat sciatic nerve against ischemia/reperfusion injury

doi: 10.3892/etm.2016.3148

Figure Lengend Snippet: Effects of MK-801 on TNF-α and TACE mRNA expression levels in the rat sciatic nerve (SN) following ischemia/reperfusion (I/R) injury. TNF-α and TACE mRNA expression levels were determined using reverse transcription-quantitative polymerase chain reaction and are expressed relative to β-actin. Agarose gel images showing TNF-α and TACE mRNA expression levels in the SN homogenates at (A) 0, (B) 6, (C) 12, (D) 24 and (E) 72 h and (F) 7 days post-reperfusion. β-actin=280 bp; TNF-α=402 bp; TACE=624 bp. Relative (G) TNF-α and (H) TACE mRNA expression levels are presented as the mean ± standard deviation (n=6). *P<0.05, **P<0.01 vs. the sham-operated group; Δ P<0.05, ΔΔ P<0.01 vs. the I/R group. TNF-α, tumor necrosis factor-α; TACE, TNF-α-converting enzyme.

Article Snippet: Tissue slices were incubated with rabbit anti-mouse TNF-α polyclonal antibody (1:100; BA14901; Wuhan Boster Bio-Engineering Co., Ltd.) at 4°C overnight.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Standard Deviation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: TRIM38 inhibits TNFα- and IL-1β–triggered NF-κB activation by mediating lysosome-dependent degradation of TAB2/3

doi: 10.1073/pnas.1318227111

Figure Lengend Snippet: Overexpression of TRIM38 inhibits TNFα- and IL-1β–triggered signaling. (A) Effects of TRIM38 on TNFα- and IL-1β–triggered NF-κB activation in HEK293 cells. HEK293 cells (1 × 105) were transfected with the NF-κB luciferase plasmid (0.01 μg) and an HA-TRIM38 plasmid (0.2 or 0.4 μg). Twenty hours after transfection, cells were treated with TNFα (10 ng/mL) or IL-1β (10 ng/mL) or left untreated for 10 h before luciferase assays were performed. Expression of transfected TRIM38 in each unstimulated sample was examined by immunoblot analysis. (B) Effects of TRIM38 on TNFα- and IL-1β–triggered NF-κB activation in HCT116 and HeLa cells. The experiments were performed as in A. (C) Effects of TRIM38 on IFNγ-induced activation of the IRF1 promoter. The experiments were performed as in A except that the IRF1 promoter reporter plasmid was used and transfected cells were treated with IFNγ (100 ng/mL). (D) Effects of TRIM38 on TNFα- and IL-1β–induced transcription of TNFA, IL-6, and IL-8 genes. HEK293 cells were transduced with either an empty vector or an HA-TRIM38 plasmid to establish stable cell lines. Cells (4 × 105) from both stable cell lines were treated with TNFα or IL-1β for the indicated times, and then total RNA was prepared for qPCR analysis. Expression of TRIM38 in the stable cell lines was examined by immunoblot analysis (Right). (E) Effects of TRIM38 on TNFα- and IL-1β–induced cytokine of TNFα, IL-6, and IL-8. HEK293 cells were transduced with either an empty vector or an HA-TRIM38 plasmid to establish stable cell lines. Cells (4 × 105) from both stable cell lines were treated with TNFα or IL-1β for the indicated times, and then the medium was collected for ELISA analysis. (F) Effects of TRIM38 on IFNγ-induced transcription of IRF1 gene. Cells (4 × 105) were left untreated or treated with IFNγ (100 ng/mL) for the indicated times, and total RNA was extracted for qPCR analysis. Graphs show mean ± SD; n = 3. *P < 0.05; **P < 0.01.

Article Snippet: Recombinant human TNFα, IL-1β, and IFNγ (R&D Systems); mouse monoclonal antibodies against Flag (Sigma), HA (Covance), and β-actin (Sigma); mouse anti-TAK1, p-TAK1, p-IKKα/β; rabbit anti-JNK, p-JNK, p38, p-p38, Erk1/2, p-Erk1/2 (CST); rabbit anti-TRAF6, RIP1, IRAK1, TRAF2 (Santa Cruz Biotechnology); rabbit anti-TAB3 (Epitomics); LysoTracker (Invitrogene); and ELISA kits for human TNFα, IL-6, and IL-8 (BOSTER) were purchased from the indicated manufacturers.

Techniques: Over Expression, Activation Assay, Transfection, Luciferase, Plasmid Preparation, Expressing, Western Blot, Transduction, Stable Transfection, Enzyme-linked Immunosorbent Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: TRIM38 inhibits TNFα- and IL-1β–triggered NF-κB activation by mediating lysosome-dependent degradation of TAB2/3

doi: 10.1073/pnas.1318227111

Figure Lengend Snippet: Knockdown or knockout of TRIM38 potentiates TNFα- and IL-1β–triggered signaling. (A) Efficiencies of TRIM38-RNAi plasmids on TRIM38 levels. (Upper) HEK293 cells (4 × 105) were transfected with expression plasmids for TRIM38-Flag and HA-β-actin (0.1 μg each) and the indicated RNAi plasmids (1 μg each). Twenty-four hours after transfection, cell lysates were analyzed by immunoblot with anti-Flag or anti-HA. (Lower) HEK293 cells (1 × 107) were transfected with control or the indicated TRIM38-RNAi plasmids (10 μg each) for 36 h. Cell lysates were analyzed by immunoblot with anti-TRIM38 or anti–β-actin. (B) Effects of TRIM38-RNAi on TNFα- and IL-1β–triggered NF-κB activation in HEK293 and HeLa cells. The cells (1 × 105) were transfected with RNAi plasmids (1 μg each) along with the NF-κB reporter plasmid (0.01 μg). Thirty-six hours after transfection, cells were left untreated or treated with TNFα (10 ng/mL) or IL-1β (10 ng/mL) for 10 h before luciferase assays were performed. (C) Effects of TRIM38-RNAi on IFNγ-induced IRF1 promoter activation. Reporter assays were performed as in B except that cells were transfected with IRF1 promoter reporter plasmid and treated with IFNγ (100 ng/mL). (D) Effects of TRIM38 deficiency on TNFα and IL-1β–induced transcription of TNFA, IL-6, and IL-8 genes. The indicated cells (4 × 105) were treated with TNFα (10 ng/mL) or IL-1β (10 ng/mL) for the indicated times, and then total RNA was extracted for qPCR analysis. (E) Effects of TRIM38 deficiency on TNFα and IL-1β–induced cytokine production. The indicated cells (4 × 105) were treated with TNFα (10 ng/mL) or IL-1β (10 ng/mL) for the indicated times, and then the medium was collected for ELISA analysis. (F) TRIM38 deficiency potentiates TNFα-triggered MAPK activation. The indicated cells (1 × 107) were left untreated or treated with TNFα (10 ng/mL) for the indicated times. Cells were lyzed and immunoblot analysis was performed with the indicated antibodies. (G) TRIM38 deficiency potentiates IL-1β–triggered MAPK activation. The experiments were performed as in E, except that cells were treated with IL-1β (10 ng/mL). Graphs show mean ± SD; n = 3. *P < 0.05; **P < 0.01.

Article Snippet: Recombinant human TNFα, IL-1β, and IFNγ (R&D Systems); mouse monoclonal antibodies against Flag (Sigma), HA (Covance), and β-actin (Sigma); mouse anti-TAK1, p-TAK1, p-IKKα/β; rabbit anti-JNK, p-JNK, p38, p-p38, Erk1/2, p-Erk1/2 (CST); rabbit anti-TRAF6, RIP1, IRAK1, TRAF2 (Santa Cruz Biotechnology); rabbit anti-TAB3 (Epitomics); LysoTracker (Invitrogene); and ELISA kits for human TNFα, IL-6, and IL-8 (BOSTER) were purchased from the indicated manufacturers.

Techniques: Knockdown, Knock-Out, Transfection, Expressing, Western Blot, Control, Activation Assay, Plasmid Preparation, Luciferase, Enzyme-linked Immunosorbent Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: TRIM38 inhibits TNFα- and IL-1β–triggered NF-κB activation by mediating lysosome-dependent degradation of TAB2/3

doi: 10.1073/pnas.1318227111

Figure Lengend Snippet: TRIM38 interacts with and destabilizes TAB2 through its C-terminal PRY-SPRY domain. (A) TRIM38 interacts with TAB2 and TAB3 in mammalian overexpression system. HEK293 cells (1 × 107) were transfected with the indicated plasmids for 24 h. Coimmunoprecipitation and immunoblots were performed with the indicated antibodies. (B) Endogenous TRIM38 interacts with TAB2/3. HEK293 cells (3 × 107) were left untreated or treated with TNFα (Left) or IL-1β (Right) for the indicated times. Endogenous coimmunoprecipitation and immunoblots were performed with the indicated antibodies. (C) TRIM38 specifically destabilizes TAB2/3. HEK293 (4 × 105) cells were transfected with the indicated plasmids for 24 h, and then immunoblots were performed with the indicated antibodies. (D) Effects of TRIM38 truncation mutants on destabilization of TAB2. HEK293 (4 × 105) cells were transfected with the indicated plasmids for 24 h before immunoblots were performed with the indicated antibodies. (E) Analysis of TRIM38 expression in TRIM38−/− cells stably transduced with an empty vector (II), TRIM38-Flag (III), TRIM38(63-465)-Flag (IV), or TRIM38(290-465)-Flag (V), respectively and in TRIM38+/+ cells stably transduced with an empty vector (I). Cells (1 × 107) (I, II, III, IV, V) were harvested and lysed. Immunoblot analysis was performed with the indicated antibodies. (F) Reconstitution of TRIM38 or TRIM38 mutant (63–465) into TRIM38-deficient cells leads to down-regulation of TAB2. Cells (1 × 107) (I, II, III, IV, V) were left untreated or treated with TNFα or IL-1β for the indicated times. Immunoblot analysis was performed with the indicated antibodies.

Article Snippet: Recombinant human TNFα, IL-1β, and IFNγ (R&D Systems); mouse monoclonal antibodies against Flag (Sigma), HA (Covance), and β-actin (Sigma); mouse anti-TAK1, p-TAK1, p-IKKα/β; rabbit anti-JNK, p-JNK, p38, p-p38, Erk1/2, p-Erk1/2 (CST); rabbit anti-TRAF6, RIP1, IRAK1, TRAF2 (Santa Cruz Biotechnology); rabbit anti-TAB3 (Epitomics); LysoTracker (Invitrogene); and ELISA kits for human TNFα, IL-6, and IL-8 (BOSTER) were purchased from the indicated manufacturers.

Techniques: Over Expression, Transfection, Western Blot, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Mutagenesis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: TRIM38 inhibits TNFα- and IL-1β–triggered NF-κB activation by mediating lysosome-dependent degradation of TAB2/3

doi: 10.1073/pnas.1318227111

Figure Lengend Snippet: TRIM38 mediates lysosomal degradation of TAB2. (A) Effects of inhibitors on TRIM38-mediated destabilization of TAB2. HEK293 cells (4 × 105) were transfected with the indicated plasmids. Fourteen hours after transfection, the cells were treated with the indicated inhibitors for 6 h before immunoblot analysis was performed. (B) Effects of NH4Cl and MG132 on down-regulation of TAB2 triggered by TNFα and IL-1β. HEK293 (1 × 107) cells were treated with NH4Cl or MG132 for 4 h and then further treated with TNFα and IL-1β for 2 h before immunoblot analysis was performed. (C) TRIM38 promotes translocation of TAB2 to the lysosome. HEK293 cells (1 × 105) were transfected with Cherry-TAB2 and GFP-LAMP1 (Left) or CFP-TRIM38 (Right). Twenty hours after transfection, cells were fixed with 4% (wt/vol) paraformaldehyde and subjected for confocal microscopy. (D) Effect of TRIM38 deficiency on TNFα- or IL-1β–induced colocalization of TAB2 with the lysosomes. TRIM38+/+, TRIM38−/−, or TRIM38−/− cells reconstituted with the PRY-SPRY domain (1 × 105) were transfected with GFP-TAB2. Twenty hours after transfection, cells were stained with Red Lysotracker (200 nM) for 2 h and treated with TNFα (20 ng/mL) or IL-1β (20 ng/mL) for 1 h and then fixed with 4% (wt/vol) paraformaldehyde and subjected to confocal microscopy. A random 10 cells in each sample were used for calculating the colocalization dots that were normalized to the total lysosome-red dots. Graphs show mean ± SD; n = 3. **P < 0.01.

Article Snippet: Recombinant human TNFα, IL-1β, and IFNγ (R&D Systems); mouse monoclonal antibodies against Flag (Sigma), HA (Covance), and β-actin (Sigma); mouse anti-TAK1, p-TAK1, p-IKKα/β; rabbit anti-JNK, p-JNK, p38, p-p38, Erk1/2, p-Erk1/2 (CST); rabbit anti-TRAF6, RIP1, IRAK1, TRAF2 (Santa Cruz Biotechnology); rabbit anti-TAB3 (Epitomics); LysoTracker (Invitrogene); and ELISA kits for human TNFα, IL-6, and IL-8 (BOSTER) were purchased from the indicated manufacturers.

Techniques: Transfection, Western Blot, Translocation Assay, Confocal Microscopy, Staining

Journal: Drug Design, Development and Therapy

Article Title: Total saponin of Dioscoreae hypoglaucae rhizoma ameliorates streptozotocin-induced diabetic nephropathy

doi: 10.2147/DDDT.S99670

Figure Lengend Snippet: Effect of TSD on renal TGF-β1, CTGF, TNF-α, IL-1β, and IL-6 levels in diabetic rats

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α, interleukin-1β (IL-1β), interleukin-6 (IL-6), TGF-β1, and connective tissue growth factor (CTGF) were obtained from Cusabio Biotech Co. Ltd, Wuhan, People’s Republic of China.

Techniques: