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: Infection and Immunity

Article Title: Phagocytic Receptors Activate Syk and Src Signaling during Borrelia burgdorferi Phagocytosis

doi: 10.1128/IAI.00004-17

Figure Lengend Snippet: Src family and Syk kinases have a role in phagocytosis and signaling in response to B. burgdorferi. (A) Wild-type BMDMs were pretreated with Src-1, PP1 (Src family kinase inhibitor), and piceatannol (Syk inhibitor) for 30 min prior to the addition of B. burgdorferi at an MOI of 10. Cells were stimulated for 6 h, supernatants were collected, and the concentrations of secreted IL-6 and TNF-α were determined by an ELISA. Data are shown as means and standard errors of the means of results from three independent experiments. *, P < 0.05. (B) Wild-type BMDMs were stimulated with Bb-GFP for 60 min at 37°C. Any inhibitors or blocking agents used were added for 30 min prior to Bb-GFP stimulation. Following Bb-GFP stimulation, cells were fixed in 1% paraformaldehyde and stained in 2% goat serum and 1% saponin. Noninternalized B. burgdorferi bacteria were stained with anti-B. burgdorferi antibodies followed by a secondary anti-rabbit Alexa Fluor 594 antibody. Phagocytosis is shown as percent internalized bacteria per cell. *, P < 0.05. (C) Wild-type BMDMs were stimulated with B. burgdorferi at an MOI of 10 for 60 min. Cells were lysed and enriched for phosphoproteins. Samples from the input pool and after phosphoenrichment were run on an SDS-PAGE gel and blotted with antibodies to Syk and Src. Shown is a representative blot from three independent experiments.

Article Snippet: Total Syk antibody (Cell Signaling), total Src family kinase antibody (Cell Signaling), and total FAK antibody (Cell Signaling) were used at 1:1,000 dilutions in a 5% bovine serum albumin (BSA) block.

Techniques: Enzyme-linked Immunosorbent Assay, Blocking Assay, Staining, SDS Page

Journal: Infection and Immunity

Article Title: Phagocytic Receptors Activate Syk and Src Signaling during Borrelia burgdorferi Phagocytosis

doi: 10.1128/IAI.00004-17

Figure Lengend Snippet: Both integrin β1 and MARCO activate Src family kinases in response to B. burgdorferi. (A) Wild-type (WT) and MARCO-deficient (M−/−) BMDMs were stimulated with B. burgdorferi at an MOI of 10 for 60 min (+ Bb) or the control (No Bb). Cells were lysed and enriched for phosphoproteins. Samples from the input pool and after phosphoenrichment were run on SDS-PAGE gels and blotted with antibodies to SFKs. Shown is a representative blot from three independent experiments. Densitometry was performed by using ImageJ, and data from three independent experiments are shown as mean values (± standard deviations) of the intensities of protein bands of interest normalized to the input. *, P < 0.05. (B) GD25 integrin β1-null MEFs (β1−/−) and integrin β1-reconstituted GD25 MEFs (+β1) were stimulated with B. burgdorferi at an MOI of 10 for 60 min or the control. Cells were lysed and enriched for phosphoproteins. Samples from the input pool and after phosphoenrichment were run on SDS-PAGE gels and blotted with antibodies to SFKs. Shown is a representative blot from three independent experiments. Densitometry was performed by using ImageJ, and data from three independent experiments are shown as mean values (± standard deviations) of the intensities of protein bands of interest normalized to the input. *, P < 0.05. (C) Wild-type and MARCO-deficient BMDMs were stimulated with B. burgdorferi at an MOI of 10 for 60 min. Cells were lysed and enriched for phosphoproteins. Samples from the input pool and after phosphoenrichment were run on SDS-PAGE gels and blotted with antibodies to FAK. Shown is a representative blot from three independent experiments. Densitometry was performed by using ImageJ, and data from three independent experiments are shown as mean values (± standard deviations) of the intensities of protein bands of interest normalized to the input. *, P < 0.05.

Article Snippet: Total Syk antibody (Cell Signaling), total Src family kinase antibody (Cell Signaling), and total FAK antibody (Cell Signaling) were used at 1:1,000 dilutions in a 5% bovine serum albumin (BSA) block.

Techniques: SDS Page