Journal: Molecular Pain

Article Title: Neuropeptide deficient mice have attenuated nociceptive, vascular, and inflammatory changes in a tibia fracture model of complex regional pain syndrome

doi: 10.1186/1744-8069-8-85

Figure Lengend Snippet: Determination of hindpaw skin cytokine and NGF levels at 3 weeks after fracture (FX) in WT, SP deficient (SP KO, Tac1 −/− ), and CGRP receptor deficient (RAMP1 KO, RAMP1 −/− ) mice. TNFα (A, E), IL-1β (B, F), IL-6 (C, G) and NGF (D, H) production in hindpaw skin were determined by Bio-Rad bead suspension array (for cytokines) and EIA (for NGF). Baseline cytokine and NGF levels were the same in all 3 groups of mice. Tibia fracture induced a significant increase in all three cytokine levels and NGF production in WT FX group vs WT Controls, however, fracture did not induce increased TNFα ( A ), IL-1β ( B ), or NGF ( D ) levels in SP KO FX mice compared to unfractured SP KO Control mice. The IL-6 ( C ) levels were elevated in the SP KO FX mice, compared to unfractured SP KO Control mice, but the IL-6 increase the SP KO FX mice was considerable reduced compared to the IL-6 levels in the WT FX mice. A similar pattern was observed in the RAMP1 KO mice for TNFα ( E ), IL-1β ( F ), or NGF ( H ), except the increase in IL-6 ( G ) levels in the RAMP1 KO FX mice was even greater than the increase observed in the WT FX mice. Data are expressed as mean values (pg/mg protein) ± SE (n = 8 per group). *P < 0.05, **P < 0.001, and ***P < 0.0001 vs the respective unfractured Control mice; #P < 0.05, ##P < 0.001, and ###P < 0.0001 vs WT FX mice.

Article Snippet: For IL-6 immunostaining rabbit anti human IL-6 antibody (1:400, LifeSpan Biosciences Inc) was labeled with donkey anti-rabbit IgG (1:500) conjugated with Dylight 488 secondary antibodies (Jackson ImmunoResearch).

Techniques:

Journal: Molecular Pain

Article Title: Neuropeptide deficient mice have attenuated nociceptive, vascular, and inflammatory changes in a tibia fracture model of complex regional pain syndrome

doi: 10.1186/1744-8069-8-85

Figure Lengend Snippet: Fluorescence photomicrographs of keratin (red), IL-1β (green), and IL-6 (green) immunostaining in the plantar hindpaw skin at 3 weeks post-fracture (FX). The top two rows of panels show keratinocyte and IL-1β immunostaining from a wildtype (WT) unfractured control (Cont) mouse (first row) and the second row panels are from a WT fracture mouse. The bottom two rows of panels show keratinocyte and IL-6 immunostaining from a WT unfractured control mouse (third row) and the bottom row panels are from a WT fracture mouse. Double labeling demonstrates fracture-induced IL-1β and IL-6 protein up-regulation localized within the epidermal keratinocyte layer of the WT mice. Scale bar = 20 μm.

Article Snippet: For IL-6 immunostaining rabbit anti human IL-6 antibody (1:400, LifeSpan Biosciences Inc) was labeled with donkey anti-rabbit IgG (1:500) conjugated with Dylight 488 secondary antibodies (Jackson ImmunoResearch).

Techniques: Fluorescence, Immunostaining, Labeling

Journal: Molecular Pain

Article Title: Neuropeptide deficient mice have attenuated nociceptive, vascular, and inflammatory changes in a tibia fracture model of complex regional pain syndrome

doi: 10.1186/1744-8069-8-85

Figure Lengend Snippet: After baseline testing, WT mice underwent a right distal tibia fracture (FX) and the hindlimb was casted for 3 weeks, then the cast was removed and the next day the animals retested (n = 12 per cohort). Then the FX mice were either subcutaneously injected with the IL-6 receptor antagonist TB-2-081 (FX + TB-2-081, n = 8) or vehicle (FX + Vehicle, n = 6). A control cohort of WT mice did not undergo tibia fracture (Control, n = 12). At 15 minutes post-injection the mice were retested. The IL-6 receptor antagonist partially inhibited the development of von Frey allodynia ( A ) and hindlimb unweighting ( B ) in the fracture mice, when compared to the extent of allodynia and unweighting observed in the vehicle injected mice after fracture. The IL-6 receptor antagonist failed to reverse the hindpaw warmth ( C ) and edema ( D ) that was observed in the vehicle injected fracture mice. ***P < 0.001 vs Control, ##P < 0.01, and ###P < 0.001 vs FX + Vehicle.

Article Snippet: For IL-6 immunostaining rabbit anti human IL-6 antibody (1:400, LifeSpan Biosciences Inc) was labeled with donkey anti-rabbit IgG (1:500) conjugated with Dylight 488 secondary antibodies (Jackson ImmunoResearch).

Techniques: Injection

Journal: Oncotarget

Article Title: Hyperinsulinemia enhances interleukin-17-induced inflammation to promote prostate cancer development in obese mice through inhibiting glycogen synthase kinase 3-mediated phosphorylation and degradation of interleukin-17 receptor

doi: 10.18632/oncotarget.7296

Figure Lengend Snippet: ( A – D ) 293 cells were co-transfected with Flag-IL-17RA, V5-IL-17RC, and HA-Act1, labeled with 32 P orthophosphate, and treated with 20 ng/ml IL-17A for 20 min; IP with anti-HA; autoradiography (A) followed by IB (B-D). ( E ) Anti-Flag IP followed by IB. ( F – G ) Co-IP of Flag-IL-17RA and HA-GSK3β. ( H ) 293 cells were co-transfected with Flag-IL-17RA, WT HA-GSK3β or kinase-dead mutant HA-GSK3βK85A, labeled with 32 P orthophosphate, or treated with 20 mM LiCl for 2 h; autoradiography ( 32 P) followed by IB. WE, whole cell extract.

Article Snippet: The antibodies and peptides used are as follows: rabbit anti-human phospho-IL-17RA (T780) polyclonal antibodies (B1, B2, B3, and B4), phosphorylated antigen peptide (C-LTDPH(pT)PYEEEQ) and non-phosphorylated antigen peptide (C-LTDPHTPYEEEQ) were produced through a contract by AbMART (Shanghai) CO., LTD., Shanghai, China.

Techniques: Transfection, Labeling, Autoradiography, Co-Immunoprecipitation Assay, Mutagenesis

Journal: Oncotarget

Article Title: Hyperinsulinemia enhances interleukin-17-induced inflammation to promote prostate cancer development in obese mice through inhibiting glycogen synthase kinase 3-mediated phosphorylation and degradation of interleukin-17 receptor

doi: 10.18632/oncotarget.7296

Figure Lengend Snippet: ( A – B ) 293 cells were transfected with Flag-IL-17RA and its mutants and labeled with 32 P orthophosphate; autoradiography ( 32 P) followed by IB. ( C ) IL-17RA peptide phosphorylated at T780 (p-Peptide) was treated with CIP, while IL-17RA peptide with wild-type T780 (Peptide) or with mutant T780A (mut-Peptide) were treated with recombinant GSK3, followed with dot blot analysis using B4 antibodies. ( D – E ) Flag-IL-17RA, Flag-IL-17RAT780A mutant, or empty vector was transfected into 293 cells; HeLa cells were not transfected; IP with anti-Flag, B4 (+), or control IgG (−), followed by IB; *indicates endogenous P-IL-17RA.

Article Snippet: The antibodies and peptides used are as follows: rabbit anti-human phospho-IL-17RA (T780) polyclonal antibodies (B1, B2, B3, and B4), phosphorylated antigen peptide (C-LTDPH(pT)PYEEEQ) and non-phosphorylated antigen peptide (C-LTDPHTPYEEEQ) were produced through a contract by AbMART (Shanghai) CO., LTD., Shanghai, China.

Techniques: Transfection, Labeling, Autoradiography, Mutagenesis, Recombinant, Dot Blot, Plasmid Preparation, Control

Journal: Oncotarget

Article Title: Hyperinsulinemia enhances interleukin-17-induced inflammation to promote prostate cancer development in obese mice through inhibiting glycogen synthase kinase 3-mediated phosphorylation and degradation of interleukin-17 receptor

doi: 10.18632/oncotarget.7296

Figure Lengend Snippet: ( A ) and ( D ) 239 cells stably expressing Flag-IL-17RA (293-IL-17RA cell line) and HeLa cells were treated with 2 μM AZD5363 (a pan-Akt inhibitor) and/or 50 ng/ml insulin for the indicated time periods; Western blot analysis was performed for the indicated proteins; exogenous, IL-17RA transfected into 293 cells; endogenous, endogenous IL-17RA expressed in 293 and HeLa cells. ( B ) and ( C ) 293-IL-17RA cells were treated with 20 ng/ml IL-17A, with or without 2 μM AZD5363 and/or 50 ng/ml insulin for 2 h; IL-17-downstream gene expression was determined using qRT-PCR analysis; * P < 0.05 compared to each single treatment group; ** P < 0.05 compared to the combined insulin and IL-17 treatment group (one-way ANOVA). ( E ) and ( F ) HeLa cells were treated with 20 ng/ml IL-17A, with or without 2 μM AZD5363 and/or 50 ng/ml insulin for 2 h; IL-17-downstream gene expression was determined using qRT-PCR analysis; * P < 0.05 compared to each single treatment group; ** P < 0.05 compared to the combined insulin and IL-17 treatment group (one-way ANOVA). Data represent the mean ± standard deviation of three independent experiments ( n = 3).

Article Snippet: The antibodies and peptides used are as follows: rabbit anti-human phospho-IL-17RA (T780) polyclonal antibodies (B1, B2, B3, and B4), phosphorylated antigen peptide (C-LTDPH(pT)PYEEEQ) and non-phosphorylated antigen peptide (C-LTDPHTPYEEEQ) were produced through a contract by AbMART (Shanghai) CO., LTD., Shanghai, China.

Techniques: Stable Transfection, Expressing, Western Blot, Transfection, Gene Expression, Quantitative RT-PCR, Standard Deviation

Journal: Oncotarget

Article Title: Hyperinsulinemia enhances interleukin-17-induced inflammation to promote prostate cancer development in obese mice through inhibiting glycogen synthase kinase 3-mediated phosphorylation and degradation of interleukin-17 receptor

doi: 10.18632/oncotarget.7296

Figure Lengend Snippet: ( A – D ) 293 cells stably expressing Flag-IL-17RA were treated with 20 mM LiCl or 10 μM MG132, and/or 50 μg/ml CHX; P-IL-17RA was detected using B4 and total exogenous IL-17RA was detected using anti-Flag; the levels of total exogenous IL-17RA (C) were normalized by the levels of GAPDH (D); exo, exogenously transfected IL-17RA; endo, endogenously expressed IL-17RA; * P < 0.05 compared to LiCl and MG132 treated groups (Student's t test). ( E ) Flag-IL-17RA and Flag-IL-17RA-T780A mutant were co-transfected with HA-tagged ubiquitin (HA-Ub), with or without 10 μM MG132 treatment; non-specific, an unknown band detected by anti-HA antibodies, which was not IL-17RA or IgG heavy chain (see ). ( F ) Flag-IL-17RA and Flag-IL-17RA-T780A mutant were co-transfected with HA-tagged ubiquitin (HA-Ub) or K48-only ubiquitin (HA-K48), with 10 μM MG132 treatment, followed by IP and IB.

Article Snippet: The antibodies and peptides used are as follows: rabbit anti-human phospho-IL-17RA (T780) polyclonal antibodies (B1, B2, B3, and B4), phosphorylated antigen peptide (C-LTDPH(pT)PYEEEQ) and non-phosphorylated antigen peptide (C-LTDPHTPYEEEQ) were produced through a contract by AbMART (Shanghai) CO., LTD., Shanghai, China.

Techniques: Stable Transfection, Expressing, Transfection, Mutagenesis, Ubiquitin Proteomics

Journal: Oncotarget

Article Title: Hyperinsulinemia enhances interleukin-17-induced inflammation to promote prostate cancer development in obese mice through inhibiting glycogen synthase kinase 3-mediated phosphorylation and degradation of interleukin-17 receptor

doi: 10.18632/oncotarget.7296

Figure Lengend Snippet: ( A – I ) Human normal prostate, PIN, and prostate cancer tissues were double stained for P-IL-17RA using B4 (arrowheads) and for basal cells using anti-p63 (arrows). ( J ) Quantification of P-IL-17RA staining.

Article Snippet: The antibodies and peptides used are as follows: rabbit anti-human phospho-IL-17RA (T780) polyclonal antibodies (B1, B2, B3, and B4), phosphorylated antigen peptide (C-LTDPH(pT)PYEEEQ) and non-phosphorylated antigen peptide (C-LTDPHTPYEEEQ) were produced through a contract by AbMART (Shanghai) CO., LTD., Shanghai, China.

Techniques: Staining