Journal: Immunity

Article Title: Intestinal permeability and IgA provoke immune vasculitis linked to cardiovascular inflammation

doi: 10.1016/j.immuni.2019.05.021

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Lamina Propria Dissociation Kit, mouse , Miltenyi Biotec , Cat# 130–097-410.

Techniques: Recombinant, Flow Cytometry, Modification, Staining, Enzyme-linked Immunosorbent Assay, Software, Microscopy

Journal: Nature Communications

Article Title: B cells inhibit bone formation in rheumatoid arthritis by suppressing osteoblast differentiation

doi: 10.1038/s41467-018-07626-8

Figure Lengend Snippet: B cells inhibit osteoblast differentiation in vitro by secreting CCL3 and TNF. a – c BM B cells from WT or TNF-Tg mice were purified, as in Fig. . B cells were co-cultured with MPCs ± anti-CCL3 neutralizing Ab ± anti-TNF neutralizing Ab in OB-inducing medium for 2 days. a ALP+ area was measured. * p < 0.05 vs. WT, # p < 0.05 vs. TNF-Tg treated with IgG. ( b , c ) B cells were removed and protein lysates from the MPCs were subjected to Western blot analyses. Expression levels of NF-κB ( b ) and ERK signaling ( c ) molecules in cell lysates from MPCs were assessed. Supplementary Fig. shows uncropped gel images. Each experiment was performed 3 to 5 times. Representative images and quantifications shown in the figure come from one independent experiment. All error bars represent s.e.m. One way ANOVA followed by Dunnett’s post-hoc multiple comparisons was performed

Article Snippet: After multinucleated cells were observed under a microscope, the cells were fixed, stained for TRAP activity to identify OCs (TRAP+ cells containing >3 nuclei) and counted . (6) For blocking experiments with neutralizing Abs, anti-mouse CCL3 Ab (AF-450-NA), anti-mouse TNF Ab (AF-410-NA), anti-human CCL3 Ab (MAB270), and anti-human TNF Ab (MAB610), were purchased from R & D systems and used according to the manufacturer’s instructions. (7) For human B cell stimulation, purified B cells were cultured with 5 μg/ml CpG 2006 (Oligos Etc.) plus 10 μg/ml Anti-Ig (A+G+M) (109-006-064, Jackson ImmunoResearch Laboratories) or PBS control for 4 h. At the end of the culture period, conditioned medium was collected for co-culture or ELISA.

Techniques: In Vitro, Purification, Cell Culture, Western Blot, Expressing

Journal: Nature Communications

Article Title: B cells inhibit bone formation in rheumatoid arthritis by suppressing osteoblast differentiation

doi: 10.1038/s41467-018-07626-8

Figure Lengend Snippet: RA B cells express high levels of CCL3 and TNF and inhibit osteoblast bone formation in vivo. a MPCs were treated with different doses of CCL3 in OB-inducing medium for 2 days. The area of ALP+ cells was measured. * p < 0.05 vs. Veh. b Expression levels of Runx2 and ALP in 50 ng/ml CCL3 treated cells from a were measured by qPCR. Fold-changes were calculated by dividing the values with those from Veh. * p < 0.05 vs. Veh. c CCL3+ B cells (yellow) were detected in subchondral bone (SB) area in frozen sections of proximal tibiae from TNF-Tg mice and their WT littermates by double IF staining with anti-B220 (red) and anti-CCL3 (green) Abs. Bar = 25 μm. d , e BM B cells from WT or TNF-Tg mice were purified and stimulated (S) with 2.5 μg/ml anti-CD40 Ab +10 ng/ml IL4 +10 μg/ml LPS or vehicle (U) for 4 h. CCL3 ( d ) and TNF ( e ) protein expression levels were assessed in the culture media by ELISA. f – h WT MPCs were transplanted with BM B cells from WT, TNF-Tg, TNF-Tg/CCL3-KO, TNF-Tg/TNF-KO, and TNF-Tg/CCL3-KO/TNF-KO mice by subcutaneous surgical implantation into recipient SCID mice. 4 weeks later, the implants were harvested and H&E staining ( f ) and Goldner’s Trichrome ( g ) staining were performed. B bone. G GelFoam. Bar = 50 μm. h A histomorphometric analysis of bone volume to tissue volume in H&E-stained sections. i MPCs from GFP transgenic mice were transplanted with BM B cells from mTmG mice. Representative images show mTmG+ B cells (red) and GFP+ MPCs (green) in the implants. White dashed lines indicate the bone surface. Bar = 25 μm. * p < 0.05 as indicated groups. Each experiment was performed 3 to 5 times. Representative images and quantifications are shown from one independent experiment. All error bars represent s.e.m. Two-tailed unpaired Student’s t -test was performed for b . One way ANOVA followed by Dunnett’s post-hoc multiple comparisons was performed for all the others

Article Snippet: After multinucleated cells were observed under a microscope, the cells were fixed, stained for TRAP activity to identify OCs (TRAP+ cells containing >3 nuclei) and counted . (6) For blocking experiments with neutralizing Abs, anti-mouse CCL3 Ab (AF-450-NA), anti-mouse TNF Ab (AF-410-NA), anti-human CCL3 Ab (MAB270), and anti-human TNF Ab (MAB610), were purchased from R & D systems and used according to the manufacturer’s instructions. (7) For human B cell stimulation, purified B cells were cultured with 5 μg/ml CpG 2006 (Oligos Etc.) plus 10 μg/ml Anti-Ig (A+G+M) (109-006-064, Jackson ImmunoResearch Laboratories) or PBS control for 4 h. At the end of the culture period, conditioned medium was collected for co-culture or ELISA.

Techniques: In Vivo, Expressing, Staining, Purification, Enzyme-linked Immunosorbent Assay, Transgenic Assay, Two Tailed Test

Journal: Nature Communications

Article Title: B cells inhibit bone formation in rheumatoid arthritis by suppressing osteoblast differentiation

doi: 10.1038/s41467-018-07626-8

Figure Lengend Snippet: B cells from RA patients inhibit OB differentiation. a–c Peripheral blood (PB) B cells were purified, as in Fig. and stimulated (S) with CpG2006+anti-Ig (A+G+M) Ab or vehicle (U) for 4 h. a CCL3 and TNF mRNA expression was detected by qPCR. * p < 0.05. b CCL3 and TNF protein expression levels were assessed in the culture media by ELISA. * p < 0.05. c Conditioned medium (40% by volume) from B cell culture were co-cultured with human MSCs ± anti-CCL3 neutralizing Ab ± anti-TNF neutralizing Ab in OB-inducing medium for 3 days. ALP+ area was measured. * p < 0.05 vs. U, # p < 0.05 vs. S-IgG. d PB B cells from RA patients and healthy controls (HC) were co-cultured with human MSCs in OB-inducing medium for 3 days. ALP staining was performed. * p < 0.05 vs. HC. e The expression levels of ALP and Runx2 in hMSCs in d were measured by qPCR. Fold-changes were calculated by dividing patient values by the value from HC cells. Values represent individual HC/RA. *p < 0.05 vs. HC. f RA synovium was stained with Abs to CD20 (B cells), CCL3 and TNF. White arrows indicate the cells with dual staining. Bar = 25 μm. 3 or 6 patients and their controls were included in each experiment. All of these experiments were repeated at least once with representative images and quantifications shown. All error bars represent s.e.m. One way ANOVA followed by Dunnett’s post-hoc multiple comparisons was performed for c . Two-tailed unpaired Student’s t -test was performed for all the others

Article Snippet: After multinucleated cells were observed under a microscope, the cells were fixed, stained for TRAP activity to identify OCs (TRAP+ cells containing >3 nuclei) and counted . (6) For blocking experiments with neutralizing Abs, anti-mouse CCL3 Ab (AF-450-NA), anti-mouse TNF Ab (AF-410-NA), anti-human CCL3 Ab (MAB270), and anti-human TNF Ab (MAB610), were purchased from R & D systems and used according to the manufacturer’s instructions. (7) For human B cell stimulation, purified B cells were cultured with 5 μg/ml CpG 2006 (Oligos Etc.) plus 10 μg/ml Anti-Ig (A+G+M) (109-006-064, Jackson ImmunoResearch Laboratories) or PBS control for 4 h. At the end of the culture period, conditioned medium was collected for co-culture or ELISA.

Techniques: Purification, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Staining, Two Tailed Test

Journal: Neuro-Oncology

Article Title: Systemic AAV9-IFNβ gene delivery treats highly invasive glioblastoma

doi: 10.1093/neuonc/now097

Figure Lengend Snippet: Peripheral expression of hIFNβ contributes to the therapeutic effect of systemically infused scAAV9/CB-hIFNβ in the GBM8 mouse model. (A) scAAV9/CB-hIFNβ (CB) or scAAV9/CB-hIFNβ-miRBS-1-122 (de-targeted) vectors were infused at the doses shown or phosphate-buffered saline (PBS) at 2 weeks after tumor implantation and plasma hIFNβ levels measured 1 month later by ELISA (n = 4 per group). Columns represent mean values, and error bars indicate SD. ****P < .0001 in unpaired 2-tailed t test. (B) Tumor-associated bioluminescence signal (TABS) kinetics are represented as fold change over signal at 1 week after tumor implantation for all treatment groups. CB vector, de-targeted vector, or scAAV9/TBG-hIFNβ (TBG vector) were injected 2 weeks after tumor implantation at doses shown. Data are shown as mean ± SD. (C) Kaplan-Meier survival curves for different treatment groups. In log-rank test, **P < .01; ns, not significant (P > .05). (D–F) Comparison of hIFNβ mRNA levels in brain, liver, and skeletal muscle in mice from different treatment groups at the humane or study endpoints by RT-qPCR (n = 3 per group). Top panels show the comparative delta CT values, and bottom panels show relative expression. Data are shown as mean ± SD. In unpaired 2 tailed t test, *P < .05; **P < .01; ***P < .001; Abbreviation: ns, not significant (P > .05).

Article Snippet: Quantification of Human IFNβ in Cell-conditioned Media and Mouse Plasma hIFNβ in conditioned growth medium and mouse plasma was measured using ELISA assays (41 410 and 41 415, respectively; PBL Assay Science).

Techniques: Expressing, Saline, Tumor Implantation, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Injection, Comparison, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Ubiquitination of RIPK1 regulates its activation mediated by TNFR1 and TLRs signaling in distinct manners

doi: 10.1038/s41467-020-19935-y

Figure Lengend Snippet: a , b Ripk1 +/+ and Ripk1 K612R/K612R immortalized MEFs were pretreated with SM-164 (100 nM) or 5Z-7 (5Z 7-oxozeanol) (200 nM) for 1.5 h in the presence or absence of Nec-1s (10 μM) as indicated, and then treated with TNFα (100 ng/ml)/Z-VAD (25 μM) ( a ) or TNFα (100 ng/ml) ( b ) for different time points. Cell survival was measured by Cell TiterGlo. Data are presented as mean ± SEM of n = 3 biologically independent samples. Two-way ANOVA with Bonferroni’s multiple comparison test. c WT and RIPK1-K612R primary BMDMs were pretreated with SM-164 (100 nM) or 5Z-7 (200 nM) for 1.5 h in the presence or absence of Nec-1s (10 μM) or GSK’872 (10 μM) as indicated, and then treated with TNFα (100 ng/ml) alone or Z-VAD (25 μM) alone or Z-VAD (25 μM) plus TNFα (100 ng/ml) for 10 h. Cell survival was measured by Cell TiterGlo. Data are presented as mean ± SEM of n = 3 biologically independent samples. Two-way ANOVA with Bonferroni’s multiple comparison test.

Article Snippet: The sources of the materials used in this study: human recombinant soluble TNFα (Novoprotein Scientific), mouse recombinant soluble TNFα (R&D Systems), flag mouse TNFα (homemade), 5Z-7-Oxozeaenol (Sigma-Aldrich), GSK’872 (Calbiochem), Z-VAD.fmk (Selleckchem), R-7-Cl-O-Nec-1 (Nec-1s), and SM-164 (custom synthesized).

Techniques: Comparison

Journal: Nature Communications

Article Title: Ubiquitination of RIPK1 regulates its activation mediated by TNFR1 and TLRs signaling in distinct manners

doi: 10.1038/s41467-020-19935-y

Figure Lengend Snippet: a Expected and observed frequency of genotypes in the offspring as adult from intercrosses of Ripk1 +/K612R mice. b Body weight of 8-weeks-old male Ripk1 +/+ ( n = 8) and Ripk1 K612R/K612R ( n = 8) mice, and female Ripk1 +/+ ( n = 6) and Ripk1 K612R/K612R ( n = 6) mice. Data are presented as mean ± SEM of n = 8 or 6 biologically independent mice. Unpaired two-tailed Student’s t - test. c , d Western blotting analysis of RIPK1 protein levels in the spleen and thymus ( c ) and intestine ( d ) from indicated mice at different ages. Uncropped blots in Source Data. e IL-6 and TNFα levels in the sera of Ripk1 +/+ ( n = 8) and Ripk K612R/K612 ( n = 8) (8–9 weeks-old) mice determined by ELISA. Data are presented as mean ± SEM of n = 8 biologically independent mice. Unpaired two-tailed Student’s t -test. f Representative images of large intestine and colon length of Ripk1 +/+ ( n = 4) and Ripk1 K612R/K612R mice ( n = 4, 8 weeks old). Data are presented as Mean±SEM of n = 4 biologically independent mice. Unpaired two-tailed Student’s t -test. g Haematoxylin & Eosin staining for general histology, PAS/AB staining for goblet cells and immunohistochemical staining of F4/80, GR-1 and Ki67 in the sections of colon from Ripk1 +/+ ( n = 4) and Ripk1 K612R/K612R ( n = 4) mice (8–9 weeks old) (Scale bars, 50 μm). h QRT-PCR analysis of cytokine and chemokine expression in the colon of Ripk1 +/+ ( n = 8) and Ripk1 K612R/K612R ( n = 8) (8–9 weeks old). Data are presented as mean ± SEM of n = 8 biologically independent mice. Unpaired two-tailed Student’s t -test. i QRT-PCR analysis of cFLIP expression in the colon of Ripk1 +/+ ( n = 6) and Ripk1 K612R/K612R ( n = 6) (8–9 weeks old). Data are presented as mean ± SEM of n = 6 biologically independent mice. Unpaired two-tailed Student’s t -test. j Representative images of spleens and spleen weight of Ripk1 +/+ mice ( n = 3) and Ripk1 K612R/K612R mice ( n = 3) (8–9 weeks old). k The populations of CD45 + CD11b + GR-1 + cells in the spleens of Ripk1 +/+ ( n = 6) and Ripk1 K612R/K612R ( n = 6) mice, and bone marrow of Ripk1 +/+ ( n = 5) and Ripk1 K612R/K612R ( n = 5) mice (8 weeks old). Dot plot analysis was performed to visualize the percentages of indicated cell populations. Data are presented as mean ± SEM biologically independent mice. Unpaired two-tailed Student’s t -test.

Article Snippet: The sources of the materials used in this study: human recombinant soluble TNFα (Novoprotein Scientific), mouse recombinant soluble TNFα (R&D Systems), flag mouse TNFα (homemade), 5Z-7-Oxozeaenol (Sigma-Aldrich), GSK’872 (Calbiochem), Z-VAD.fmk (Selleckchem), R-7-Cl-O-Nec-1 (Nec-1s), and SM-164 (custom synthesized).

Techniques: Two Tailed Test, Western Blot, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemical staining, Quantitative RT-PCR, Expressing

Journal: Nature Communications

Article Title: Ubiquitination of RIPK1 regulates its activation mediated by TNFR1 and TLRs signaling in distinct manners

doi: 10.1038/s41467-020-19935-y

Figure Lengend Snippet: a Body weight of 8 weeks-old male Ripk1 +/+ mice treated with antibiotics ( n = 6) or vehicle ( n = 6), and male Ripk1 K612R/K612R mice treated with antibiotics ( n = 6) or vehicle ( n = 6) for 4 weeks. Data are presented as mean ± SEM of n = 6 biologically independent mice. One-way ANOVA with Dunnett’s multiple comparison test. b IL-6 and TNFα level in sera of 8 weeks-old Ripk1 +/+ mice treated with antibiotics ( n = 7) or vehicle ( n = 7), and Ripk1 K612R/K612R mice treated with antibiotics ( n = 7) or vehicle ( n = 7) for 4 weeks. Data are presented as Mean±SEM of n = 7 biologically independent mice. One-way ANOVA with Dunnett’s multiple comparison test. c Representative images of spleens and spleen weight of 8–9 weeks-old Ripk1 +/+ mice treated with antibiotics ( n = 6) or vehicle ( n = 6), and Ripk1 K612R/K612R mice treated with antibiotics ( n = 6) or vehicle ( n = 6) for 4 weeks. Data are presented as Mean±SEM of n = 8 or 7 biologically independent mice. One-way ANOVA with Dunnett’s multiple comparison test. d Representative images of the large intestine and colon length of 8 weeks-old male Ripk1 +/+ mice treated with antibiotics ( n = 4) or vehicle ( n = 4), and male Ripk1 K612R/K612R mice treated with antibiotics ( n = 4) or vehicle ( n = 4) for 4 weeks. Data are presented as mean ± SEM of n = 4 biologically independent mice. One-way ANOVA with Dunnett’s multiple comparison test. e Western blotting analysis of RIPK1 protein levels of the colon and spleen from indicated mice treated with antibiotics or vehicle for 4 weeks as in (d). Uncropped blots in the Source Data file. f Haematoxylin & Eosin staining, PAS/AB staining, and immunohistochemical staining of F4/80, GR-1, and Ki67 in the sections of colon from 8-weeks-old Ripk1 +/+ mice treated with antibiotics ( n = 3) or vehicle ( n = 3), and Ripk1 K612R/K612R mice treated with antibiotics ( n = 3) or vehicle ( n = 3) for 4 weeks (Scale bars, 50 μm). g QRT-PCR analysis of cytokine and cFLIP expression in the colon of 8-weeks-old Ripk1 +/+ mice treated with antibiotics ( n = 8 or 6) or vehicle ( n = 8), and Ripk1 K612R/K612R mice treated with antibiotics (n = 8) or vehicle ( n = 8) for 4 weeks. Data are presented as mean ± SEM of n = 8 or 6 biologically independent mice. One-way ANOVA with Dunnett’s multiple comparison test. h The populations of CD45 + CD11b + GR-1 + cells in the spleens and bone marrow of 8-weeks-old Ripk1 +/+ mice treated with antibiotics ( n = 6) or vehicle ( n = 6), and Ripk1 K612R/K612R mice treated with antibiotics ( n = 6) or vehicle ( n = 4) for 4 weeks. Dot plot analysis was performed to visualize the percentages of indicated cell populations. Data are presented as mean ± SEM of n = 6 or 4 biologically independent mice. One-way ANOVA with Dunnett’s multiple comparison test.

Article Snippet: The sources of the materials used in this study: human recombinant soluble TNFα (Novoprotein Scientific), mouse recombinant soluble TNFα (R&D Systems), flag mouse TNFα (homemade), 5Z-7-Oxozeaenol (Sigma-Aldrich), GSK’872 (Calbiochem), Z-VAD.fmk (Selleckchem), R-7-Cl-O-Nec-1 (Nec-1s), and SM-164 (custom synthesized).

Techniques: Comparison, Western Blot, Staining, Immunohistochemical staining, Quantitative RT-PCR, Expressing

Journal: Nature Communications

Article Title: Ubiquitination of RIPK1 regulates its activation mediated by TNFR1 and TLRs signaling in distinct manners

doi: 10.1038/s41467-020-19935-y

Figure Lengend Snippet: a Primary Ripk1 +/+ and Ripk1 K612R/K612R BMDMs were pretreated with Nec-1s (10 μM), GSK’872 (10 μM) or vehicle in the presence or absence of Z-VAD (25 μM) for 30 min respectively, and then treated with Poly (I:C) (20 μg/ml) or LPS (50 ng/ml) as indicated for 10 h. Cell survival was measured by CellTiterGlo. Data are presented as mean ± SEM of n = 3 biologically independent samples. Two-way ANOVA with Bonferroni’s multiple comparison test. b , c Complex IIb was isolated by RIPK3 antibody and analyzed by western blotting in primary BMDMs isolated from indicated mouse strains pretreated with Z-VAD (25 μM) or vehicle for 30 min, and then treated with LPS (50 ng/ml) for indicated time points. Uncropped blots in the Source Data file. d Quantification of IL-1β and TNFα in the cultural supernatant from primary Ripk1 +/+ and Ripk1 K612R/K612R BMDMs treated with LPS (50 ng/ml) for indicated time points by ELISA. e Western blotting analysis of the cell lysates and cultural supernatant of Ripk1 +/+ and Ripk1 K612R/K612R primary BMDMs treated with LPS (100 ng/ml) for indicated time points. Uncropped blots in the Source Data file. f Primary BMDMs isolated from indicated mouse strains were treated with LPS (50 ng/ml) plus Z-VAD (25 μM) or Poly (I:C) (20 μg/ml) or plus Z-VAD (25 μM) as indicated for 10 h. Cell survival was measured by Cell TiterGlo. Data are presented as Mean±SEM of n = 3 biologically independent samples. Two-way ANOVA with Bonferroni’s multiple comparison test. g Western blotting analysis of primary BMDMs isolated from indicated mouse strains were treated with LPS (50 ng/ml) plus Z-VAD (25 μM) for indicated time points. Uncropped blots in the Source Data file. h , i Quantification of IL-1β in cultural supernatant by ELISA ( h ) and western blotting analysis of cell lysates and cultural supernatant ( i ) of primary BMDMs with indicated genotypes treated with LPS(50 ng/ml) for indicated time points. Uncropped blots in the Source Data file.

Article Snippet: The sources of the materials used in this study: human recombinant soluble TNFα (Novoprotein Scientific), mouse recombinant soluble TNFα (R&D Systems), flag mouse TNFα (homemade), 5Z-7-Oxozeaenol (Sigma-Aldrich), GSK’872 (Calbiochem), Z-VAD.fmk (Selleckchem), R-7-Cl-O-Nec-1 (Nec-1s), and SM-164 (custom synthesized).

Techniques: Comparison, Isolation, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Ubiquitination of RIPK1 regulates its activation mediated by TNFR1 and TLRs signaling in distinct manners

doi: 10.1038/s41467-020-19935-y

Figure Lengend Snippet: In TNFα-stimulated WT cells, RIPK1 is rapidly recruited to TNFR1 by direct binding to the death domain (DD) of TNFR1 or that of TRADD to initiate the formation of complex I. In complex I the ubiquitination of RIPK1 is modulated by multiple E3 ubiquitin ligases such as cIAP1/2 and LUBAC complex. Ubiquitination of RIPK1 mediates NF-κB activation. Ubiquitination of RIPK1 K612 modulates overall patterns of RIPK1 ubiquitination and promotes RIPK1-RIPK1 and RIPK1-TNFR1 interaction mediated by its DD, which leads to its activation and interaction with FADD/Caspase-8 to mediate RDA or with RIPK3 to mediate necroptosis when caspase-8 is inactivated. K612R mutation reduces the recruitment of RIPK1 and inhibits the activation of RIPK1 upon stimulation of TNFR1 by TNFα. RIPK1 K612R mutation inhibits cell death induced by TNFα through disrupting RIPK1 dimerization and RIPK1-TNFR1 interaction mediated by DD domain. In LPS or Poly (I:C)-stimulated WT cells, RIPK1 and RIPK3 interact with TRIF which regulates the activation of RIPK1 and RIPK3. Ubiquitination of RIPK1 K612 promotes RIPK1 ubiquitination, dimerization and interaction of RIPK1 with FADD, which in turn recruits FADD to suppress necroptosis and caspase-1 activation by restricting RIPK3 activation in response to TLR3 and TLR4. RIPK1 K612R mutation disrupts the interaction of RIPK1 and FADD to promote RIPK3-dependent necroptosis and inflammation induced by LPS or Poly(I:C).

Article Snippet: The sources of the materials used in this study: human recombinant soluble TNFα (Novoprotein Scientific), mouse recombinant soluble TNFα (R&D Systems), flag mouse TNFα (homemade), 5Z-7-Oxozeaenol (Sigma-Aldrich), GSK’872 (Calbiochem), Z-VAD.fmk (Selleckchem), R-7-Cl-O-Nec-1 (Nec-1s), and SM-164 (custom synthesized).

Techniques: Binding Assay, Ubiquitin Proteomics, Activation Assay, Mutagenesis

Journal: Molecular medicine reports

Article Title: Beneficial effects of the traditional medicine Igongsan and its constituent ergosterol on dextran sulfate sodium-induced colitis in mice.

doi: 10.3892/mmr.2015.3824

Figure Lengend Snippet: Figure 2. Effects of IGS on the levels of TNF‑α and IL‑6 in colon tissue from DSS‑treated mice. Experimental colitis was induced in the mice by supplementing their drinking water with 5% (w/v) DSS for 7 days. IGS (100 mg/kg/day) was administered once a day for 7 days prior to intake of 5% DSS. At the end of the experiment, the colon tissue was harvested and homogenized. (A and B) Levels of TNF‑α and IL‑6 in the indicated groups were measured by ELISA. Values are expressed as the mean ± standard error of the mean (n=6) of duplicate determinations from three separate experiments #P<0.05 vs. control; *P<0.05 vs. DSS alone. IGS, Igongsan; DSS, dextran sodium sulfate; SFZ, sulfasalazine; TNF, tumor necrosis factor; IL‑6, interleukin‑6.

Article Snippet: Mouse TNF-α affinity purified polyclonal, goat IgG (cat no. AF‐410‐NA), mouse TNF-α biotinylated affinity purified polyclonal, goat IgG (cat no. BF410) and recombinant mouse TNF‐α (cat no. 410-MT) were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Control