Journal: Annals of Translational Medicine

Article Title: Isorhamnetin inhibits amplification of influenza A H1N1 virus inflammation mediated by interferon via the RIG-I/JNK pathway

doi: 10.21037/atm-21-3532

Figure Lengend Snippet: Antibody information

Article Snippet: The enzyme-linked immunosorbent assay (ELISA) kit was purchased from Multi-sciences (Lianke Biotech, Co., Ltd., Hangzhou, Zhejiang, China) [IL-6, EK106P; interferon inducible protein (IP)-10, EK168P; monocyte chemotactic protein (MCP)-1, EK187P]. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Source/isotype Manufacturer Art. no. Molecular weight (kDa) COX-2 Rabbit CST #12282 74 phospho-JNK Rabbit CST #4668 46/54 JNK Rabbit CST #9252 46/54 phospho-p38 Rabbit CST #4511 38 p38 Rabbit CST #9212 38 RIG-I Rabbit CST #3743 102 β-actin Rabbit CST #2118 37 Open in a separate window caption a8 Antibody information

Techniques: Molecular Weight

Journal: Annals of Translational Medicine

Article Title: Isorhamnetin inhibits amplification of influenza A H1N1 virus inflammation mediated by interferon via the RIG-I/JNK pathway

doi: 10.21037/atm-21-3532

Figure Lengend Snippet: Isorhamnetin inhibits phosphorylation of JNK, p38 MAPK proteins and expression of RIG-I. After allowing 2 h for H1N1 absorption, H1N1-infected A549 cells were treated with or without the indicated concentrations of isorhamnetin for 24 h. (A) The gene expression levels of RIG-I in H1N1-infected A549 cells treated with or without isorhamnetin were determined using qRT-PCR at 24 h. (B) Expression of RIG-I/JNK and p38 MAPK signals in the H1N1-infected A549 cells. Cells were examined by western blotting for the expression of RIG-I, P-JNK, JNK, P-p38, p38 24 h post infection. (C) The band intensities of RIG-I, P-JNK and P-p38 were semiquantified using imageJ (normalized to the loading control GAPDH). The data are presented as the mean ± SEM (n=3). *P<0.05, **P<0.01 and ***P<0.001. qRT-PCR, quantitative real-time polymerase chain reaction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; SEM, standard error of the mean.

Article Snippet: The enzyme-linked immunosorbent assay (ELISA) kit was purchased from Multi-sciences (Lianke Biotech, Co., Ltd., Hangzhou, Zhejiang, China) [IL-6, EK106P; interferon inducible protein (IP)-10, EK168P; monocyte chemotactic protein (MCP)-1, EK187P]. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Source/isotype Manufacturer Art. no. Molecular weight (kDa) COX-2 Rabbit CST #12282 74 phospho-JNK Rabbit CST #4668 46/54 JNK Rabbit CST #9252 46/54 phospho-p38 Rabbit CST #4511 38 p38 Rabbit CST #9212 38 RIG-I Rabbit CST #3743 102 β-actin Rabbit CST #2118 37 Open in a separate window caption a8 Antibody information

Techniques: Phospho-proteomics, Expressing, Infection, Gene Expression, Quantitative RT-PCR, Western Blot, Control, Real-time Polymerase Chain Reaction

Journal: Annals of Translational Medicine

Article Title: Isorhamnetin inhibits amplification of influenza A H1N1 virus inflammation mediated by interferon via the RIG-I/JNK pathway

doi: 10.21037/atm-21-3532

Figure Lengend Snippet: Isorhamnetin inhibited the inflammatory response of IFN-β-pretreated IAV infected cells via the RIG-I/JNK pathway. (A) Expression of RIG-I/JNK and p38 MAPK signals in IAV (H1N1)-infected A549 cells pretreated with IFN-β. Cells were examined by western blotting for the expression of RIG-I, phosphorylated-JNK, JNK, phosphorylated-p38, and p38 24 h post infection; (B) the band intensities of RIG-I, phosphorylated-JNK and phosphorylated-p38 were semiquantified using imageJ (normalized to the loading control GAPDH). The data are presented as the mean ± SEM (n=3). ***P<0.001. IFN-β, interferon-β; IAV, influenza A virus; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; SEM, standard error of the mean.

Article Snippet: The enzyme-linked immunosorbent assay (ELISA) kit was purchased from Multi-sciences (Lianke Biotech, Co., Ltd., Hangzhou, Zhejiang, China) [IL-6, EK106P; interferon inducible protein (IP)-10, EK168P; monocyte chemotactic protein (MCP)-1, EK187P]. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Source/isotype Manufacturer Art. no. Molecular weight (kDa) COX-2 Rabbit CST #12282 74 phospho-JNK Rabbit CST #4668 46/54 JNK Rabbit CST #9252 46/54 phospho-p38 Rabbit CST #4511 38 p38 Rabbit CST #9212 38 RIG-I Rabbit CST #3743 102 β-actin Rabbit CST #2118 37 Open in a separate window caption a8 Antibody information

Techniques: Infection, Expressing, Western Blot, Control, Virus

Journal: Annals of Translational Medicine

Article Title: Isorhamnetin inhibits amplification of influenza A H1N1 virus inflammation mediated by interferon via the RIG-I/JNK pathway

doi: 10.21037/atm-21-3532

Figure Lengend Snippet: Diagrammatic sketch showing the mechanism by which isorhamnetin attenuates IAV (H1N1)-induced proinflammatory responses and injury. After infection, influenza virus is recognized by RIG-I, which induces the production of type I IFN, cytokines, and chemokines. The IFN-α and IFN-β act on the IFN receptors (IFNARs) of adjacent cells via autocrine and paracrine activity to activate the MAPK pathway. This pathway is a cascade of protein kinases that is triggered by specific extracellular signals such as type I IFN. Upon extracellular stimulation, MAPKs are activated downstream of sequentially activated protein kinases, MAPK kinase kinase (MKKK) and MAPK kinase (MKK). Subsequently, JNK and p38, 2 MAPK family proteins, become continuously activated (i.e., phosphorylated) by multiple upstream kinases in the MAPK pathway, thus inducing the expression of pro-inflammatory and IFN. Following, IFN interacts with IFNAR in the form of paracrine or autocrine to cause positive feedback, accompanied by excessive release of pro-inflammatory cytokines. The inhibition of RIG-I signaling by isorhamnetin attenuates RIG-I-linked proinflammatory IFN production, which results in a reduction in JNK activation and thus a decrease in the amplification of proinflammatory responses. IAV, influenza A virus; IFN, interferon.

Article Snippet: The enzyme-linked immunosorbent assay (ELISA) kit was purchased from Multi-sciences (Lianke Biotech, Co., Ltd., Hangzhou, Zhejiang, China) [IL-6, EK106P; interferon inducible protein (IP)-10, EK168P; monocyte chemotactic protein (MCP)-1, EK187P]. table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Source/isotype Manufacturer Art. no. Molecular weight (kDa) COX-2 Rabbit CST #12282 74 phospho-JNK Rabbit CST #4668 46/54 JNK Rabbit CST #9252 46/54 phospho-p38 Rabbit CST #4511 38 p38 Rabbit CST #9212 38 RIG-I Rabbit CST #3743 102 β-actin Rabbit CST #2118 37 Open in a separate window caption a8 Antibody information

Techniques: Infection, Virus, Activity Assay, Expressing, Inhibition, Activation Assay, Amplification

Journal: Cells

Article Title: Arachidonic Acid Cascade and Eicosanoid Production Are Elevated While LTC4 Synthase Modulates the Lipidomics Profile in the Brain of the HIVgp120-Transgenic Mouse Model of NeuroHIV

doi: 10.3390/cells11132123

Figure Lengend Snippet: MAPK signaling pathway in female LTC4SKO mice shows the reduced activity levels of p38 and ERK in protein brain lysates. The quantification of Western blot densitometries of: phosphorylated p38 MAPK (pp38)/total p38/tubulin (p38) ( A ); phosphorylated ERK1/2 (pERK)/total ERK1/2/tubulin (ERK) ( B ); and phosphorylated JNK1/2 (pJNK)/total JNK1/2/tubulin (JNK) ( C ) all normalized to their respective wild type control. Representative images of the blots are shown with male and female samples separated by a protein ladder lane are for p38 ( D ), ERK1/2 ( E ), and JNK1/2 ( F ). Male ( blue ) and female ( red ) data sets are shown, separated by a dashed line. Values in graphs are mean ± s.e.m.; n = 4–6 per genotype for both male and female data sets, 12 months of age. Individual data points are represented in the graphs by an ⚪ symbol. ANOVA and Fisher’s PLSD post hoc test for males and females were analyzed independently while normalized to their respective WT controls; p -value level of significance is as follows: * p ≤ 0.05, @@ p ≤ 0.01, @@@ p ≤ 0.001—while the p -value significance correlations are symbolized as follows: * indicates significances from the WT, @ indicates significances from HIVgp120tg.

Article Snippet: Antibodies phospho-p38 (1°Ab- 1:1000; anti-rabbit 2°Ab- 1:5000, 43 kDa) (Cell Signaling, Danvers, MA, USA; 9211), total p38 (1°Ab- 1:2000; anti-rabbit 2°Ab- 1:25,000, 40 kDa) (Cell Signaling; 9212), phospho-ERK1 (1°Ab- 1:1000; anti-rabbit 2°Ab- 1:5000, 42/44 kDa) (Cell Signaling; 9101), total ERK1 (1°Ab- 1:2000; anti-rabbit 2°Ab- 1:5000, 42/44 kDa) (Cell Signaling; 9102), active JNK (1°Ab- 1:1000; anti-rabbit 2°Ab- 1:3000, 46/54 kDa) (Promega, Madison, WI, USA; V793A), total JNK (1°Ab- 1:1000; anti-rabbit 2°Ab- 1:3000, 46/54 kDa) (Cell Signaling; 9252), and α-tubulin (1°Ab- 1:2000; anti-mouse 2°Ab- 1:10,000, 50 kDa) (Sigma-Aldrich, Burlington, MA, USA; T9026).

Techniques: Activity Assay, Western Blot

Journal: Investigative Ophthalmology & Visual Science

Article Title: Defining the Role of Mitochondrial Fission in Corneal Myofibroblast Differentiation

doi: 10.1167/iovs.63.4.2

Figure Lengend Snippet: Intracellular mediators underlying anti-fibrotic effects of Mdivi-1. ( A ) Representative Western blot depicting phospho- and total-SMAD3 expression levels in protein extracts from cultured cat corneal fibroblasts. Cells were incubated ± 10 µM Mdivi-1, ± 1 ng/mL TGF-β1 and finally 2.3 µM SB431542 with 1 ng/mL TGF-β1, as indicated. ( B ) Plot of densitometric ratios for phospho-SMAD3 expression relative to total-SMAD2/3, normalized to the TGF-β1 treatment. One-way repeated measures ANOVA: F(3,11) = 116.3, P < 0.0001. Significances from post-hoc Tukey's HSD tests relative to TGF-β1 alone are denoted: * P < 0.05, # P < 0.01. ( C ) Representative Western blots of corneal fibroblast extracts for phosphor-p54 and -p46 and total-JNK expression under the conditions indicated. ( D ) Plot of densitometric ratios for p-p54 JNK expression relative to total-p54 JNK, normalized to the TGF-β1 treatment. One-way repeated measures ANOVA: F(3,11) = 7.48, P = 0.01884. Significances relative to the TGF-β1 condition were computed and are denoted as in B . ( E ) Representative Western blot of corneal fibroblast extracts showing phospho-p38, phospho-ERK, and phospho-AKT expression levels under the conditions indicated. ( F ) Plot of densitometry ratios for phosphorylated proteins relative to β-actin, normalized to the TGF-β1 treatment. One-way repeated measures ANOVAs demonstrated significant effects of treatments for all 3 molecules: p38 F(3,11) = 37.52, P = 0.0003; pERK F(3,11) = 11.12, P = 0.0073; pAKT F(3,11) = 8.85, P = 0.0127. Significances from post-hoc Tukey's HSD tests relative to TGF-β1 are indicated as in B and D . For all graphs, data shown are means ± SD from 3 experimental replicates.

Article Snippet: Blots were incubated overnight at 4°C containing primary antibodies to the following targets at the dilutions indicated: Type 1 collagen (COL1; 1:2000; no. LF-68, kindly provided by Dr. Larry W. Fisher, NIH, Bethesda, MD, USA), total Fibronectin (t-FN; 1:2000; #H-300; Santa Cruz Inc.), α-SMA (1:10,000; no. MA5-11547; Thermo Fisher Scientific, Waltham, MA, USA), total SMAD 2/3 (1:2000; no. 8685; Cell Signaling Technology, Danvers, MA, USA), phosphorylated small mothers against decapentaplegic 3 (SMAD3) (S423+S425; 1:1000; no. P00059-1; Boster Technology, Pleasanton, CA, USA), phospho-p46/54 JNK Thr183/Tyr185 (1:1000; no. 4671; Cell Signaling Technology), phospho-p38 MAPK Thr180/Tyr182 (1:1000; no. 9215; Cell Signaling Technology), phospho-p44/42 MAPK Thr202/Tyr204 (1:1000; no. 9106; Cell Signaling Technology), phospho-AKT Ser473 (1:1000; no. 4060; Cell Signaling Technology), total-p46/54 JNK (1:1000; no. 9252; Cell Signaling Technology), p-Drp1 Ser616 (1:500; no. 3455; Cell Signaling Technology), total-Drp1 (1:1000; no. 611112; BD Biosciences, San Jose, CA, USA), Opa1 (1:1000; no. 612606; BD Biosciences), Mfn1 (1:1000; ab57602; Abcam, Boston, MA, USA), Mfn2 (1:1000; no. M6319; Sigma Aldrich), and β-actin-HRP (1:5000; no. sc-47778; Santa Cruz Inc.).

Techniques: Western Blot, Expressing, Cell Culture, Incubation