Journal: International journal of molecular medicine

Article Title: 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol, a metabolite of ginsenoside Rb1, enhances the production of hyaluronic acid through the activation of ERK and Akt mediated by Src tyrosin kinase in human keratinocytes.

doi: 10.3892/ijmm.2015.2121

Figure Lengend Snippet: Figure 3. 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol (20GPPD) induces the phosphorylation of extracellular signal-regulated kinase (ERK) and Akt in HaCaT cells. (A and B) 20GPPD induced ERK phosphorylation. (A) Cells were starved in serum-free medium and treated with 20GPPD (1 µM) for the indicated periods of time. (B) Cells were starved in serum-free medium and treated with the indicated concentrations of 20GPPD for 1 h. (C and D) 20GPPD induced Akt phosphorylation. (C) Cells were starved in serum-free medium and treated with 20GPPD (1 µM) for indicated periods of time. (D) Cells were starved in serum-free medium and treated with the indicated concentrations of 20GPPD for 1 h. The level of ERK, Akt, phosphorylated ERK (p-ERK) and phosphorylated Akt (p-Akt) was determined by immunoblot analysis as described in the Materials and methods.

Article Snippet: Antibodies against phosphorylated Src (Tyr416; 2101), Akt (Ser473; 9271), and total Akt (9272) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Phospho-proteomics, Western Blot

Journal: International journal of molecular medicine

Article Title: 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol, a metabolite of ginsenoside Rb1, enhances the production of hyaluronic acid through the activation of ERK and Akt mediated by Src tyrosin kinase in human keratinocytes.

doi: 10.3892/ijmm.2015.2121

Figure Lengend Snippet: Figure 5. Src is the upstream regulator of extracellular signal-regulated kinase (ERK) and Akt in mediating the 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol (20GPPD)-induced expression of hyaluronic acid (HA). (A) 20GPPD induced Src phosphorylation in HaCaT cells. Cells were starved in serum-free medium and treated with the indicated concentrations of 20GPPD for 20 min. (B and C) Cells were pre-treated with PP2 (Src inhibitor) for 1 h, and then exposed to 20GPPD for an additional 3 h. (B) HA concentrations were measured by enzyme-linked immunosorbent assay (ELISA) and (C) the expression of hyaluronan synthase 2 (HAS2) and β-actin was determined by immunoblot analysis. Data represent the means ± SD. ##P<0.01 vs. the untreated control; **P<0.01 vs. 20GPPD-treated group. (D) PP2 attenuated the 20GPPD-induced phosphorylation of ERK and Akt in HaCaT cells. Following pre-treatment with PP2 for 1 h, the cells were exposed to 1 µM 20GPPD for 1 h. Proteins were then subjected to immunoblot analysis to determine the level of ERK, phosphorylated ERK (p-ERK), Akt, and phosphorylated Akt (p-Akt) expression.

Article Snippet: Antibodies against phosphorylated Src (Tyr416; 2101), Akt (Ser473; 9271), and total Akt (9272) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Expressing, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: Stem cells (Dayton, Ohio)

Article Title: Transient c-Src Suppression During Endodermal Commitment of Human Induced Pluripotent Stem Cells Results in Abnormal Profibrotic Cholangiocyte-Like Cells

doi: 10.1002/stem.2950

Figure Lengend Snippet: Transient inhibition of Src kinase activity during endodermal commitment of human iPSCs results in biliary cells expressing markers associated with fibrosis. (A): Quantitative transcriptional expression of markers associated with fibrosis COL1, SMA, MMP2, TIMP1, TIMP2, LOXL2, PDGFC, TGFβ, YAP1, and VIM at HP stage, in control, and days 8–10 cells treated with NPP1 during endodermal commitment. LX2, a human primary hepatic stellate cell line, was used as a control. Immunofluorescence staining of a cholangiocyte marker CK7 (red) with fibrosis markers (B) COL1 (green), (C) SMA (green), and (D) YAP1 (green). DAPI (blue). Scale 100 μm. (E): Quantitative transcriptional expression of markers associated with inflammation IL-6, TNFα in control and NPP1-treated cells. (F): Protein secretion of IL-6 and TNFα in control and NPP1-treated cells evaluated by enzyme-linked immunosorbent assay. (*p < .05, **p < .01, ***p < .001). Abbreviations: COL1, collagen type 1; DAPI, 4′, 6-diamidino-2-phenylindole; DP, ductal progenitor; FGF4, fibroblast growth factor 4; HGF, hepatocyte growth factor; HP, hepatic progenitor; IL-6, interleukin-6; iPSC, induced pluripotent stem cell; NPP1, 1-naphthyl-PP1; OSM, oncostatin M; SMA, smooth muscle actin; YAP1, yes-associated protein 1.

Article Snippet: Enzyme-Linked Immunosorbent Assay Total cellular protein was extracted and sandwich enzyme-linked immunosorbent assay (ELISA) for phosphorylated Src (Tyr416; Cell Signaling Technology #7953C) was performed by following manufacturer’s protocol.

Techniques: Inhibition, Activity Assay, Expressing, Immunofluorescence, Staining, Marker, Enzyme-linked Immunosorbent Assay

Journal: PLoS Pathogens

Article Title: Prion protein signaling induces M2 macrophage polarization and protects from lethal influenza infection in mice

doi: 10.1371/journal.ppat.1008823

Figure Lengend Snippet: ( A ) Western blotting (left panel) for total and phosphorylated SFK in the lungs from control IgG- and 38–2 mAb-treated mice at 3 dpi with 200 IFU of IAV/PR8. Actb, β-actin. The percent density of total SFK (middle panel) and SFK phosphorylated at tyrosine 416 (right panel) against the densities of the corresponding proteins in IgG-treated, infected lungs after being normalized by the densities of β-actin. **, p<0.01. ( B ) Survival rate (%, upper panels) and body weight loss (%, lower panels) of WT mice intraperitoneally administrated with control IgG (left panels) and 38–2 mAb (right panel) together with 20 mg/mouse of DS 1 day before intranasal infection with 200 IFU of IAV/PR8. Error bars, SD. ( C ) Western blotting (upper panel) for total SFK, phosphorylated SFK (Tyr416), the cleaved caspase 3, and the viral protein M2 in the lungs from control IgG/DMSO-, 38–2 mAb/DMSO-, control IgG/DS-, and 38–2 mAb/DS-treated mice at 3 dpi with 200 IFU of IAV/PR8. Actb, β-actin. Lungs from uninfected, saline/DMSO-treated mice were used as a control. The percent density (lower panels) of total SFK, phosphorylated SFK (Tyr416), the cleaved caspase 3, and the viral protein M2 against the densities of the corresponding proteins in uninfected, saline/DMSO-treated lungs after being normalized by the densities of β-actin. *, p<0.05; **, p<0.01.

Article Snippet: Anti-podoplanin antibody (Cat. No. D190-3) and anti-β-actin antibody (Cat. No. M177-3) from MBL (Nagoya, Japan;), anti-SP-C (Cat. No. sc-7706) and anti-CC10 antibodies (Cat. No. sc-9772) from Santa Cruz Biotechnology (Santa Cruz, CA), anti-pro-caspase 3 (Cat. No. 9662S), anti-cleaved caspase 3 (Cat. No. 9664S), anti-total SFK (Cat. No. 2109S) and anti-phosphorylated SFK (Tyr416) antibodies (Cat. No. 2101S) from Cell Signaling (Beverly, MA), anti-PB1 (Cat. No. GTX125923), anti-NS1 (Cat. No. GTX125990), anti-M2 (Cat. No. GTX125951) and anti-NP (Cat. No. GTX125989) antibodies from GeneTex (Irvine, CA), anti-SOD1 antibody (Cat. No. ab13498) from Abcam (Cambridge, UK), anti-CD3 (Cat. No. MAB4841), anti-MGL1/2 (Cat. No. AF4297) and HRP -conjugated anti-goat IgG antibodies (Cat. No. HAF017) from R&D systems (Minneapolis, MN), anti-MPO antibody from Proteintech (Rosemont, IL; Cat. No. 66177-1-Ig), PE anti-mouse/human CD11b antibody (Cat. No. 101207) from Biolegend (San Diego, CA), vitality full length hrGFP polyclonal antibody (Cat. No. 240141) from Agilent (Santa Clara, CA), HRP-conjugated anti-mouse IgG antibodies (Cat. No. NA931), HRP-conjugated anti-rabbit IgG antibodies (Cat. No. NA934), HRP-conjugated anti-rat IgG antibodies (Cat. No. NA935) from GE Healthcare (Buckinghamshire, UK), Alexa Fluor 596 donkey anti-mouse IgG antibody (Cat. No. A21203), Alexa Fluor 488 goat anti-rabbit IgG antibody (Cat. No. A11008), Alexa Fluor 596 donkey anti-goat IgG antibody (Cat. No. A11058), Texas Red-X goat anti-rat IgG antibody (Cat. No. T6392), and Alexa Fluor 647 goat anti-mouse antibody (Cat. No. A28181) from Invitrogen (Carlsbad, CA).

Techniques: Western Blot, Control, Infection, Saline

Journal: PLoS Pathogens

Article Title: Prion protein signaling induces M2 macrophage polarization and protects from lethal influenza infection in mice

doi: 10.1371/journal.ppat.1008823

Figure Lengend Snippet: ( A ) Double immunofluorescent staining for phosphorylated SFK (Tyr416) together with MGL1/2, CD3, and MPO in the lungs from control IgG- and 38–2 mAb-treated mice uninfected and at 3 dpi with 200 IFU of IAV/PR8. Un, uninfected. Bar, 200 μ m. ( B ) Fold mRNA expression (2 -ΔCt ) analyzed by real-time RT-PCR for IL-6, TNF-α, IFN-α, INF-γ, MCP-1, iNOS, IFIT1, MxA, ARG1, MGL1, and IL-10 in the lungs from control IgG/DMSO, control IgG/DS-, 38–2 mAb/DMSO-, and 38–2 mAb/DS-treated mice uninfected and at 3 dpi with 200 IFU of IAV/PR8 (n = 3 in each group). Un, uninfected.*, p<0.05;**, p<0.01.

Article Snippet: Anti-podoplanin antibody (Cat. No. D190-3) and anti-β-actin antibody (Cat. No. M177-3) from MBL (Nagoya, Japan;), anti-SP-C (Cat. No. sc-7706) and anti-CC10 antibodies (Cat. No. sc-9772) from Santa Cruz Biotechnology (Santa Cruz, CA), anti-pro-caspase 3 (Cat. No. 9662S), anti-cleaved caspase 3 (Cat. No. 9664S), anti-total SFK (Cat. No. 2109S) and anti-phosphorylated SFK (Tyr416) antibodies (Cat. No. 2101S) from Cell Signaling (Beverly, MA), anti-PB1 (Cat. No. GTX125923), anti-NS1 (Cat. No. GTX125990), anti-M2 (Cat. No. GTX125951) and anti-NP (Cat. No. GTX125989) antibodies from GeneTex (Irvine, CA), anti-SOD1 antibody (Cat. No. ab13498) from Abcam (Cambridge, UK), anti-CD3 (Cat. No. MAB4841), anti-MGL1/2 (Cat. No. AF4297) and HRP -conjugated anti-goat IgG antibodies (Cat. No. HAF017) from R&D systems (Minneapolis, MN), anti-MPO antibody from Proteintech (Rosemont, IL; Cat. No. 66177-1-Ig), PE anti-mouse/human CD11b antibody (Cat. No. 101207) from Biolegend (San Diego, CA), vitality full length hrGFP polyclonal antibody (Cat. No. 240141) from Agilent (Santa Clara, CA), HRP-conjugated anti-mouse IgG antibodies (Cat. No. NA931), HRP-conjugated anti-rabbit IgG antibodies (Cat. No. NA934), HRP-conjugated anti-rat IgG antibodies (Cat. No. NA935) from GE Healthcare (Buckinghamshire, UK), Alexa Fluor 596 donkey anti-mouse IgG antibody (Cat. No. A21203), Alexa Fluor 488 goat anti-rabbit IgG antibody (Cat. No. A11008), Alexa Fluor 596 donkey anti-goat IgG antibody (Cat. No. A11058), Texas Red-X goat anti-rat IgG antibody (Cat. No. T6392), and Alexa Fluor 647 goat anti-mouse antibody (Cat. No. A28181) from Invitrogen (Carlsbad, CA).

Techniques: Staining, Control, Expressing, Quantitative RT-PCR

Journal: PLoS Pathogens

Article Title: Prion protein signaling induces M2 macrophage polarization and protects from lethal influenza infection in mice

doi: 10.1371/journal.ppat.1008823

Figure Lengend Snippet: ( A ) Uncropped, full picture of Western blotting for PrP C with 38–2 mAb in the lungs (Lg) and peritoneal macrophages (PM) from WT and Prnp 0/0 mice. Actb, β-actin. ( B ) Western blotting for total SFK, phosphorylated SFK (Tyr416), and MGL1/2 in peritoneal macrophages from WT and Prnp 0/0 mice 3 hrs after treatment with control IgG and 38–2 mAb together with DS or control DMSO. ( C ) ELISA for INF-γ, TNF-α, IL-4, and IL-10 in the culture medium from WT peritoneal macrophages at 0.5, 4, 16, and 24 hours (hrs) after treatment with control IgG and 38–2 mAb.*, p<0.05;** p<0.01. ( D ) Western blotting for total SFK, phosphorylated SFK (Tyr416), and MGL1/2 in peritoneal macrophages (PM) from WT and Prnp 0/0 mice 1 and 3 days after peritoneal injection with control IgG and 38–2 mAb together with control DMSO and DS.

Article Snippet: Anti-podoplanin antibody (Cat. No. D190-3) and anti-β-actin antibody (Cat. No. M177-3) from MBL (Nagoya, Japan;), anti-SP-C (Cat. No. sc-7706) and anti-CC10 antibodies (Cat. No. sc-9772) from Santa Cruz Biotechnology (Santa Cruz, CA), anti-pro-caspase 3 (Cat. No. 9662S), anti-cleaved caspase 3 (Cat. No. 9664S), anti-total SFK (Cat. No. 2109S) and anti-phosphorylated SFK (Tyr416) antibodies (Cat. No. 2101S) from Cell Signaling (Beverly, MA), anti-PB1 (Cat. No. GTX125923), anti-NS1 (Cat. No. GTX125990), anti-M2 (Cat. No. GTX125951) and anti-NP (Cat. No. GTX125989) antibodies from GeneTex (Irvine, CA), anti-SOD1 antibody (Cat. No. ab13498) from Abcam (Cambridge, UK), anti-CD3 (Cat. No. MAB4841), anti-MGL1/2 (Cat. No. AF4297) and HRP -conjugated anti-goat IgG antibodies (Cat. No. HAF017) from R&D systems (Minneapolis, MN), anti-MPO antibody from Proteintech (Rosemont, IL; Cat. No. 66177-1-Ig), PE anti-mouse/human CD11b antibody (Cat. No. 101207) from Biolegend (San Diego, CA), vitality full length hrGFP polyclonal antibody (Cat. No. 240141) from Agilent (Santa Clara, CA), HRP-conjugated anti-mouse IgG antibodies (Cat. No. NA931), HRP-conjugated anti-rabbit IgG antibodies (Cat. No. NA934), HRP-conjugated anti-rat IgG antibodies (Cat. No. NA935) from GE Healthcare (Buckinghamshire, UK), Alexa Fluor 596 donkey anti-mouse IgG antibody (Cat. No. A21203), Alexa Fluor 488 goat anti-rabbit IgG antibody (Cat. No. A11008), Alexa Fluor 596 donkey anti-goat IgG antibody (Cat. No. A11058), Texas Red-X goat anti-rat IgG antibody (Cat. No. T6392), and Alexa Fluor 647 goat anti-mouse antibody (Cat. No. A28181) from Invitrogen (Carlsbad, CA).

Techniques: Western Blot, Control, Enzyme-linked Immunosorbent Assay, Injection

Journal: PLoS Pathogens

Article Title: Prion protein signaling induces M2 macrophage polarization and protects from lethal influenza infection in mice

doi: 10.1371/journal.ppat.1008823

Figure Lengend Snippet: ( A ) Western blotting for total SFK, phosphorylated SFK (Tyr416), and MGL1/2 in peritoneal macrophages from WT and Prnp 0/0 mice 3 hrs after treatment with control IgG and 3S9 and 2H9 mAbs together with control DMSO and DS. ( B ) Survival rate (%, upper panels) and body weight loss (%, lower panels) of WT mice intraperitoneally administered with control IgG and 3S9 and 2H9 mAbs 1 day before intranasal infection with 100 (left panels) or 200 IFU (right panels) of IAV/PR8. Error bars, SD. *, p<0.05; **, p<0.01; IgG vs 3S9. #, <0.05; IgG vs 2H9.

Article Snippet: Anti-podoplanin antibody (Cat. No. D190-3) and anti-β-actin antibody (Cat. No. M177-3) from MBL (Nagoya, Japan;), anti-SP-C (Cat. No. sc-7706) and anti-CC10 antibodies (Cat. No. sc-9772) from Santa Cruz Biotechnology (Santa Cruz, CA), anti-pro-caspase 3 (Cat. No. 9662S), anti-cleaved caspase 3 (Cat. No. 9664S), anti-total SFK (Cat. No. 2109S) and anti-phosphorylated SFK (Tyr416) antibodies (Cat. No. 2101S) from Cell Signaling (Beverly, MA), anti-PB1 (Cat. No. GTX125923), anti-NS1 (Cat. No. GTX125990), anti-M2 (Cat. No. GTX125951) and anti-NP (Cat. No. GTX125989) antibodies from GeneTex (Irvine, CA), anti-SOD1 antibody (Cat. No. ab13498) from Abcam (Cambridge, UK), anti-CD3 (Cat. No. MAB4841), anti-MGL1/2 (Cat. No. AF4297) and HRP -conjugated anti-goat IgG antibodies (Cat. No. HAF017) from R&D systems (Minneapolis, MN), anti-MPO antibody from Proteintech (Rosemont, IL; Cat. No. 66177-1-Ig), PE anti-mouse/human CD11b antibody (Cat. No. 101207) from Biolegend (San Diego, CA), vitality full length hrGFP polyclonal antibody (Cat. No. 240141) from Agilent (Santa Clara, CA), HRP-conjugated anti-mouse IgG antibodies (Cat. No. NA931), HRP-conjugated anti-rabbit IgG antibodies (Cat. No. NA934), HRP-conjugated anti-rat IgG antibodies (Cat. No. NA935) from GE Healthcare (Buckinghamshire, UK), Alexa Fluor 596 donkey anti-mouse IgG antibody (Cat. No. A21203), Alexa Fluor 488 goat anti-rabbit IgG antibody (Cat. No. A11008), Alexa Fluor 596 donkey anti-goat IgG antibody (Cat. No. A11058), Texas Red-X goat anti-rat IgG antibody (Cat. No. T6392), and Alexa Fluor 647 goat anti-mouse antibody (Cat. No. A28181) from Invitrogen (Carlsbad, CA).

Techniques: Western Blot, Control, Infection

Journal: Immunology and Cell Biology

Article Title: An alternative downstream translation start site in the non‐TIR adaptor Scimp enables selective amplification of CpG DNA responses in mouse macrophages

doi: 10.1111/imcb.12540

Figure Lengend Snippet: Scimp is required for sustained LPS responses and Scimp TV1 is basally, rather than LPS‐inducibly, phosphorylated. (a, b) BMMs from wild‐type (WT) and Scimp TV1 mice were treated with LPS (1 ng mL −1 ) for the indicated time points, before being washed of LPS and replated in fresh media for a further 24 h. Supernatants were collected and assessed for IL‐12p40 production by ELISA. Data (mean ± s.e.m., n = 3 or 4 mice per genotype) are combined from three or four independent experiments. (c) BMMs from WT and Scimp TV1 (TV1) mice were differentiated with either CSF‐1 (10 ng mL −1 ; lanes at left) or GM‐CSF (10 ng mL −1 ; lanes at right) for 6 days. CSF‐1‐BMMs were stimulated with LPS (10 ng mL −1 ) and IFNγ (5 ng mL −1 ) or GM‐CSF (10 ng mL −1 ) for 24 h. Total protein was collected and assessed for Scimp expression via western blot. Data are representative of two independent experiments collected from two mice per genotype. (d) BMMs from WT and Scimp TV1 mice were differentiated using CSF‐1, then primed overnight with GM‐CSF (10 ng mL −1 ). BMMs were stimulated with LPS (10 ng mL −1 ) for the indicated time points before being lysed. Collected protein was immunoprecipitated using a rabbit anti‐Scimp antibody or an isotype control (rabbit anti‐Myc tag). Immunoprecipitated samples were assessed for levels of phosphorylated Scimp (p‐Tyr) and coimmunoprecipitated TLR4 and Lyn via western blot. Data are representative of two independent experiments. (e) TLR4 and (f) phospho‐Scimp levels from d were quantified using Image Lab and displayed relative to the corresponding immunoprecipitated Scimp band (the IC background signal in control lanes was subtracted from the appropriate sample band intensity before quantifying). Data are representative of two independent experiments collected from two mice per genotype. (g) SH2 pulldown assays were used to assess LPS‐inducible phosphorylation of Y58 (Grb2‐SH2) and Y120 (SLP‐SH2) on Scimp‐V5 in RAW264.7 cells expressing Scimp‐V5 or Scimp TV1‐V5. Data are representative of three independent experiments. Statistical significance in a and b was determined using two‐way ANOVA, followed by Bonferroni’s multiple comparison test using GraphPad Prism 9 (*** P < 0.001). GM‐CSF‐BMM, granulocyte macrophage colony‐stimulating factor‐derived bone marrow‐derived macrophages; IC, isotype control; IFN, interferon; LPS, lipopolysaccharide.

Article Snippet: Lysates were passed through a 28‐gauge needle and centrifuged at 10 000 g for 15 min. 30 µL of sample was added to columns that contained 200 µg (20 µL of 10 µg per µL stock solution) of SH2 probe [SH2 domain of either human CSK, GRB2 or SLP65 conjugated to GST and bound to Pierce protein G plus agarose beads (Life Technologies) as described in Luo et al. ].

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

Journal: Immunology and Cell Biology

Article Title: An alternative downstream translation start site in the non‐TIR adaptor Scimp enables selective amplification of CpG DNA responses in mouse macrophages

doi: 10.1111/imcb.12540

Figure Lengend Snippet: Assessment of Scimp cellular localization and phosphorylation in response to CpG DNA. (a–c) Wild‐type (WT) BMMs retrovirally transduced with either an empty vector, Scimp‐V5 or Scimp TV1‐V5 expression construct. Cells were treated with 0.3 µM fluorescently labeled (Alexa‐647‐conjugated) CpG DNA (CpG) for 30 min. Cellular localization of V5‐tagged Scimp proteins and CpG DNA were assessed via immunofluorescence microscopy (anti‐V5/Scimp: green; CpG‐647: red; DAPI: blue; scale bars: 10 µm). White arrows indicate filopodia‐localized Scimp‐V5. Similar results were observed in three independent experiments in which a total of three WT C57Bl/6 mice were retrovirally transduced (1 mouse per experiment). (b) SH2 pulldown assays were used to assess CpG DNA‐inducible phosphorylation of Y58 (Grb2‐SH2) and Y96 (CSK‐SH2) on Scimp‐V5 in CSF‐1‐BMMs expressing Scimp‐V5 or Scimp TV1‐V5. Data are representative of three independent experiments and three mice per genotype. (c) CSK‐bound Scimp and Scimp TV1 bands from b were quantified and normalized to the 0‐min control of the WT. Data (mean ± s.e.m., n = 3 mice per genotype) are combined from three independent experiments. BMM, bone marrow‐derived macrophages.

Article Snippet: Lysates were passed through a 28‐gauge needle and centrifuged at 10 000 g for 15 min. 30 µL of sample was added to columns that contained 200 µg (20 µL of 10 µg per µL stock solution) of SH2 probe [SH2 domain of either human CSK, GRB2 or SLP65 conjugated to GST and bound to Pierce protein G plus agarose beads (Life Technologies) as described in Luo et al. ].

Techniques: Transduction, Plasmid Preparation, Expressing, Construct, Labeling, Immunofluorescence, Microscopy, Derivative Assay

Journal: Biomarker Insights

Article Title: Comparative Analysis of Biomarkers in Type 2 Diabetes Patients With and Without Comorbidities: Insights Into the Role of Hypertension and Cardiovascular Disease

doi: 10.1177/11772719231222111

Figure Lengend Snippet: Important features in the healthy population with the use of LDA. Abbreviations: C5a, complement component 5a; 8-OHdG, deoxyguanosine; GSH, glutathione; GSSG, oxidized glutathione; HbA1c, hemoglobin A1C; HDL, high-density lipoprotein; IL-6, interleukin-6; IL-1β, interleukin-1β; IL-10, interleukin-10; LDL, high-density lipoprotein; MCP-1, monocyte chemoattractant protein-1; MOTSc, mitochondrial protein; p66Shc, adaptor protein; TC, total cholesterol.

Article Snippet: About 450 nm is absorbance.The Human SHC-Transforming Protein 1 ELISA kit (CUSA-BIO; Flarebio Biotech LLC) was used to measure p66shc because, according to the kit’s creators, it primarily detects Shc1 and its isoform p66shc.

Techniques:

Journal: Biomarker Insights

Article Title: Comparative Analysis of Biomarkers in Type 2 Diabetes Patients With and Without Comorbidities: Insights Into the Role of Hypertension and Cardiovascular Disease

doi: 10.1177/11772719231222111

Figure Lengend Snippet: Important features in the HT and CVD population with the use of LDA. Abbreviations: C5a, complement component 5a; 8-OHdG, deoxyguanosine; GSH, glutathione; GSSG, oxidized glutathione; HbA1c, hemoglobin A1C; HDL, high-density lipoprotein; IL-6, interleukin-6; IL-1β, interleukin-1β; IL-10, interleukin-10; LDL, high-density lipoprotein; MCP-1, monocyte chemoattractant protein-1; MOTSc, mitochondrial protein; p66Shc, adaptor protein; TC, total cholesterol.

Article Snippet: About 450 nm is absorbance.The Human SHC-Transforming Protein 1 ELISA kit (CUSA-BIO; Flarebio Biotech LLC) was used to measure p66shc because, according to the kit’s creators, it primarily detects Shc1 and its isoform p66shc.

Techniques:

Journal: Biomarker Insights

Article Title: Comparative Analysis of Biomarkers in Type 2 Diabetes Patients With and Without Comorbidities: Insights Into the Role of Hypertension and Cardiovascular Disease

doi: 10.1177/11772719231222111

Figure Lengend Snippet: Important features in the HT population with the use of LDA. Abbreviations: C5a, complement component 5a; 8-OHdG, deoxyguanosine; GSH, glutathione; GSSG, oxidized glutathione; HbA1c, hemoglobin A1C; HDL, high-density lipoprotein; IL-6, interleukin-6; IL-1β, interleukin-1β; IL-10, interleukin-10; LDL, high-density lipoprotein; MCP-1, monocyte chemoattractant protein-1; MOTSc, mitochondrial protein; p66Shc, adaptor protein; TC, total cholesterol.

Article Snippet: About 450 nm is absorbance.The Human SHC-Transforming Protein 1 ELISA kit (CUSA-BIO; Flarebio Biotech LLC) was used to measure p66shc because, according to the kit’s creators, it primarily detects Shc1 and its isoform p66shc.

Techniques:

Journal: Biomarker Insights

Article Title: Comparative Analysis of Biomarkers in Type 2 Diabetes Patients With and Without Comorbidities: Insights Into the Role of Hypertension and Cardiovascular Disease

doi: 10.1177/11772719231222111

Figure Lengend Snippet: Important features in the CVD population with the use of LDA. Abbreviations: C5a, complement component 5a; 8-OHdG, deoxyguanosine; GSH, glutathione; GSSG, oxidized glutathione; HbA1c, hemoglobin A1C; HDL, high-density lipoprotein; IL-6, interleukin-6; IL-1β, interleukin-1β; IL-10, interleukin-10; LDL, high-density lipoprotein; MCP-1, monocyte chemoattractant protein-1; MOTSc, mitochondrial protein; p66Shc, adaptor protein; TC, total cholesterol.

Article Snippet: About 450 nm is absorbance.The Human SHC-Transforming Protein 1 ELISA kit (CUSA-BIO; Flarebio Biotech LLC) was used to measure p66shc because, according to the kit’s creators, it primarily detects Shc1 and its isoform p66shc.

Techniques:

Journal: iScience

Article Title: WIP1 is a novel specific target for growth hormone action

doi: 10.1016/j.isci.2023.108117

Figure Lengend Snippet: GH induces WIP1 through GHR signaling (A) Western blots of hNCC line #1 pretreated with 20 mg/mL pegvisomant (Peg) for 1 h then treated with GH (500 ng/mL) for an additional 24 h. C, untreated control. (B) Colon tissue derived from 3- and 24-month-old WT and GHR −/− male mice. Each lane represents sample analysis derived from an individual animal. (C) hNCC line #1 transduced with lentivirus expressing GH (Lenti-GH) or empty vector (Lenti-V) and analyzed 10 days later. (D) iPSC-derived human intestinal organoid lines from 3 different patients were transduced with Lenti-V or Lenti-GH and analyzed 5 weeks later. Representative blots from one human intestinal organoid line are shown. (E) hNCC line #1 transduced with lentivirus expressing control shRNA (shControl) or GH shRNA (shGH). (F) Three different human intestinal organoid lines were transduced with GFP-expressing Lenti-V or Lenti-GH and cultured for 5 weeks, then sorted for GFP-negative cells. GFP-negative cells co-existing in organoids with either Lenti-GH or Lenti-V expressing cells were analyzed. All lines exhibited similar results. Representative blots from one human intestinal organoid line are shown. Representative blots from at least 3 independent experiments are depicted. ImageJ quantifications of western blots are depicted in Figure S2 .

Article Snippet: Src shRNA (human) lentiviral particles , Santa Cruz Biotechnology , Cat# sc-29228-V.

Techniques: Western Blot, Control, Derivative Assay, Transduction, Expressing, Plasmid Preparation, shRNA, Cell Culture

Journal: iScience

Article Title: WIP1 is a novel specific target for growth hormone action

doi: 10.1016/j.isci.2023.108117

Figure Lengend Snippet: GH induces WIP1 by activating Src/AMPK (A and B) Western blots of (A) hNCC line #1 treated with 1 μM AMPK inhibitor (Compound C) for 1 h followed by GH (500 ng/mL) for an additional 24 h and (B) human intestinal organoids treated with 2 μM AMPK inhibitor and GH (500 ng/mL) for 48 h. (C–F) Western blots of hNCC line #1 was treated with (C) GH (500 ng/mL) for up to 60 min; (D) 25 μM Src inhibitor for 24 h followed by GH (500 ng/mL) for 30 min–60 min; (E) 25 μM Src inhibitor for 1 h followed by GH (500 ng/mL) for 24 h; and (F) 25 μM Src inhibitor for 24 h followed by GH (500 ng/mL) for 3 h. Untreated cells/organoids served as control (C). (G–I) Western blots of hNCC line #1 transduced with lentivirus expressing Control shRNA (shControl) or Src shRNA (shSrc) (G) analyzed 72 h after transduction; (H) treated with GH (500 ng/mL) for 3 h; and (I) treated with GH (500 ng/mL) for 24 h. ImageJ quantifications of western blots are depicted in Figure S5 .

Article Snippet: Src shRNA (human) lentiviral particles , Santa Cruz Biotechnology , Cat# sc-29228-V.

Techniques: Western Blot, Control, Transduction, Expressing, shRNA

Journal: iScience

Article Title: WIP1 is a novel specific target for growth hormone action

doi: 10.1016/j.isci.2023.108117

Figure Lengend Snippet:

Article Snippet: Src shRNA (human) lentiviral particles , Santa Cruz Biotechnology , Cat# sc-29228-V.

Techniques: Virus, Control, shRNA, Recombinant, Cell Recovery, Protease Inhibitor, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, DC Protein Assay, Extraction, Immunoprecipitation, cDNA Synthesis, Single Cell Gel Electrophoresis, Derivative Assay, Generated, Software, Imaging, Real-time Polymerase Chain Reaction, Microscopy