igm  (Thermo Fisher)

Journal: JCI Insight

Article Title: T cell Bim levels reflect responses to anti–PD-1 cancer therapy

doi: 10.1172/jci.insight.86014

Figure Lengend Snippet: PD-1 blockade decreases Bim expression induced by PD-L1 in human CD8 + T cells.

Article Snippet: To block PD-1/PD-L1 interaction-induced Bim upregulation, preactivated CD8+ T cells were incubated with 10 μg/ml of mouse anti-human PD-1 antibody (clone MIH4, eBioscience, special order for functional grade-purified version) or humanized antibody to PD-1 (Keytruda or Opdivo, Mayo Clinic Pharmacy) for 20 minutes before culture with PD-L1 fusion protein.

Techniques: Expressing

Journal: JCI Insight

Article Title: T cell Bim levels reflect responses to anti–PD-1 cancer therapy

doi: 10.1172/jci.insight.86014

Figure Lengend Snippet: Bim upregulation is associated with PD-1 expression in melanoma patients.

Article Snippet: To block PD-1/PD-L1 interaction-induced Bim upregulation, preactivated CD8+ T cells were incubated with 10 μg/ml of mouse anti-human PD-1 antibody (clone MIH4, eBioscience, special order for functional grade-purified version) or humanized antibody to PD-1 (Keytruda or Opdivo, Mayo Clinic Pharmacy) for 20 minutes before culture with PD-L1 fusion protein.

Techniques: Expressing

Journal: JCI Insight

Article Title: T cell Bim levels reflect responses to anti–PD-1 cancer therapy

doi: 10.1172/jci.insight.86014

Figure Lengend Snippet: Changes of Bim levels predict responses to anti–PD-1 therapy in melanoma patients.

Article Snippet: To block PD-1/PD-L1 interaction-induced Bim upregulation, preactivated CD8+ T cells were incubated with 10 μg/ml of mouse anti-human PD-1 antibody (clone MIH4, eBioscience, special order for functional grade-purified version) or humanized antibody to PD-1 (Keytruda or Opdivo, Mayo Clinic Pharmacy) for 20 minutes before culture with PD-L1 fusion protein.

Techniques:

Journal: JCI Insight

Article Title: T cell Bim levels reflect responses to anti–PD-1 cancer therapy

doi: 10.1172/jci.insight.86014

Figure Lengend Snippet: Bim upregulation in tumor-reactive CD8 + T cells is dependent on PD-1 and PD-L1 interaction.

Article Snippet: To block PD-1/PD-L1 interaction-induced Bim upregulation, preactivated CD8+ T cells were incubated with 10 μg/ml of mouse anti-human PD-1 antibody (clone MIH4, eBioscience, special order for functional grade-purified version) or humanized antibody to PD-1 (Keytruda or Opdivo, Mayo Clinic Pharmacy) for 20 minutes before culture with PD-L1 fusion protein.

Techniques:

Journal: PLoS Pathogens

Article Title: RIG-I Mediates the Co-Induction of Tumor Necrosis Factor and Type I Interferon Elicited by Myxoma Virus in Primary Human Macrophages

doi: 10.1371/journal.ppat.1000099

Figure Lengend Snippet: TNF and IFN-β are required for potent restriction of MV infection in primary human fibroblasts. (A) Primary human skin fibroblasts CCD-922Sk were mock-infected or infected with MV. X-gal staining was performed to visualize blue MV foci 48 h after infection. (B) CCD-922Sk fibroblasts were infected with MV in the absence or presence of TNF or IFN-β alone or TNF plus IFN-β. MV yields were determined by the standard plaque assay using BGMK cells at 48 h after infection. (C) CCD-922Sk fibroblasts were infected with the control MV (constructed with the same vector but no human TNF gene) or the recombinant MV expressing human TNF (MV-hTNF) in the absence or presence of IFN-β. The viral yields of both control MV and MV-hTNF were determined by the standard plaque assay using RK-13 cells at 48 h after infection. (D) CCD-922Sk fibroblasts were infected with EMCV in the absence or presence of IFN-β or TNF. EMCV quantities were titrated by the standard plaque assay using BHK cells at 48 h after infection. Data in (B), (C) and (D) represent mean +/− SD.

Article Snippet: The primary antibodies were obtained from the following suppliers: anti-IRF3, anti-IRF7, anti-NF-κB p65 and anti-USF2 antibodies, Santa Cruz Biotechnology; anti-human TNF and anti-human IFN-α/β antibodies, Biosource International; anti-β-actin antibody, Sigma; anti-STAT1 antibody, Transduction Laboratories.

Techniques: Infection, Staining, Plaque Assay, Construct, Plasmid Preparation, Recombinant, Expressing

Journal: PLoS Pathogens

Article Title: RIG-I Mediates the Co-Induction of Tumor Necrosis Factor and Type I Interferon Elicited by Myxoma Virus in Primary Human Macrophages

doi: 10.1371/journal.ppat.1000099

Figure Lengend Snippet: MV-elicited production of TNF and IFN-β is mediated by RIG-I in primary human macrophages. (A) pHMs were transfected with control siRNA or siRNAs targeting cytoplasmic RNA sensors RIG-I (top panel) or MDA5 (middle panel). The cells were analyzed 48 h later by RT-PCR for the indicated mRNA levels. GAPDH was used as control. (B, C) RIG-I is critically required for MV-elicited production of both TNF (B) and IFN-β (C) in pHMs. pHMs or various siRNA pHMs were mock-infected or infected with MV and the accumulation of TNF and IFN-β in the culture supernatants was assessed by ELISA 24 h post-infection. (D) RIG-I mediates cellular restriction to MV infection in pHMs. pHMs or control siRNA or RIG-I siRNA pHMs as indicated were infected with MV. MV yields were determined by the standard plaque assay using BGMK cells at 48 h after infection. Data in (B), (C) and (D) represent mean +/− SD.

Article Snippet: The primary antibodies were obtained from the following suppliers: anti-IRF3, anti-IRF7, anti-NF-κB p65 and anti-USF2 antibodies, Santa Cruz Biotechnology; anti-human TNF and anti-human IFN-α/β antibodies, Biosource International; anti-β-actin antibody, Sigma; anti-STAT1 antibody, Transduction Laboratories.

Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Infection, Enzyme-linked Immunosorbent Assay, Plaque Assay

Journal: PLoS Pathogens

Article Title: RIG-I Mediates the Co-Induction of Tumor Necrosis Factor and Type I Interferon Elicited by Myxoma Virus in Primary Human Macrophages

doi: 10.1371/journal.ppat.1000099

Figure Lengend Snippet: Primary human macrophages produce TNF and IFN-β in response to MV infection. (A, B) Primary human macrophages (pHMs), CCD-922Sk fibroblasts and primary human lymphocytes were mock-infected or infected with MV. TNF (A) and IFN-β (B) accumulation in the culture supernatants was assessed by ELISA 24 h post-infection. (C) pHMs were mock-infected or infected with MV in the absence or presence of TNF neutralizing antibody (Ab) or IFN-α/β neutralizing Ab alone or TNF Ab plus IFN-α/β Ab as specified. MV β-gal activity was determined by measuring absorbance at 420 nm and normalized to the total cellular protein levels at 48 h after infection. (D) pHMs were infected with MV or UV-inactivated MV for various times (below lanes) and total RNA was analyzed by RT-PCR for induction of TNF and IFN-β mRNA. GAPDH was used as control. Data in (A), (B) and (C) represent mean +/− SD.

Article Snippet: The primary antibodies were obtained from the following suppliers: anti-IRF3, anti-IRF7, anti-NF-κB p65 and anti-USF2 antibodies, Santa Cruz Biotechnology; anti-human TNF and anti-human IFN-α/β antibodies, Biosource International; anti-β-actin antibody, Sigma; anti-STAT1 antibody, Transduction Laboratories.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Activity Assay, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS Pathogens

Article Title: RIG-I Mediates the Co-Induction of Tumor Necrosis Factor and Type I Interferon Elicited by Myxoma Virus in Primary Human Macrophages

doi: 10.1371/journal.ppat.1000099

Figure Lengend Snippet: IRF7 is crucial for sustaining MV-elicited TNF induction in primary human macrophages. (A) Control siRNA pHMs or IRF7 siRNA pHMs were infected with MV for various times (below lanes) and the whole cell lysates were analyzed for MV-induced expression of IRF7 protein. β-actin was used as control. (B) pHMs or various siRNA pHMs were mock-infected or infected with MV. TNF protein in the culture supernatants was assessed by ELISA 24 h post-infection. (C) IRF7 expression and TNF production kinetics. Control siRNA pHMs or IRF7 siRNA pHMs were infected with MV for various times as indicated and TNF protein in the culture supernatants was assessed by ELISA. (D) IRF7 is required for sustaining TNF gene transcription activated by MV infection. Control siRNA pHMs or IRF7 siRNA pHMs were infected with MV for various times (below lanes) and the cells were analyzed by RT-PCR for the indicated TNF mRNA levels. GAPDH was used as control. Data in (B) and (C) represent mean +/− SD.

Article Snippet: The primary antibodies were obtained from the following suppliers: anti-IRF3, anti-IRF7, anti-NF-κB p65 and anti-USF2 antibodies, Santa Cruz Biotechnology; anti-human TNF and anti-human IFN-α/β antibodies, Biosource International; anti-β-actin antibody, Sigma; anti-STAT1 antibody, Transduction Laboratories.

Techniques: Infection, Expressing, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS Pathogens

Article Title: RIG-I Mediates the Co-Induction of Tumor Necrosis Factor and Type I Interferon Elicited by Myxoma Virus in Primary Human Macrophages

doi: 10.1371/journal.ppat.1000099

Figure Lengend Snippet: IRF3 is critical for triggering MV-elicited TNF induction in primary human macrophages. (A) pHMs were transfected with control siRNA or MAVS siRNA. The cells were analyzed 48 h later by RT-PCR for the indicated mRNA levels. GAPDH was used as the control. (B) Control siRNA pHMs or RIG-I siRNA or MAVS siRNA pHMs as indicated were infected with MV for various times (below lanes) and the nuclear extracts were probed for nuclear IRF3 (nuc-IRF3). Nuclear USF2 (nuc-USF2) was used as nuclear protein loading control. (C) Control siRNA pHMs or RIG-I siRNA or MAVS siRNA pHMs as indicated were infected with MV for 0 h or 10 h and were immunofluorescence-stained for IRF3 (red). Nuclear DNA was counterstained with DAPI (blue). Bars are 10 µm. (D) pHMs were transfected with control siRNA or IRF3 siRNA as indicated and were analyzed 72 h later by immunoblotting for IRF3 protein levels. β-actin was used as control. (E) pHMs or various siRNA pHMs as indicated were infected with MV for 24 h and TNF protein in the culture supernatants was assessed by ELISA. Data represent mean +/− SD. (F) Control siRNA pHMs or IRF3 siRNA pHMs were infected with MV for various times (below lanes) and the cells were analyzed by RT-PCR for the indicated TNF mRNA levels. GAPDH was used as control.

Article Snippet: The primary antibodies were obtained from the following suppliers: anti-IRF3, anti-IRF7, anti-NF-κB p65 and anti-USF2 antibodies, Santa Cruz Biotechnology; anti-human TNF and anti-human IFN-α/β antibodies, Biosource International; anti-β-actin antibody, Sigma; anti-STAT1 antibody, Transduction Laboratories.

Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Infection, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Localization of a constitutively active, phagocyte-like NADPH oxidase in rabbit aortic adventitia: Enhancement by angiotensin II

doi:

Figure Lengend Snippet: Localization of NADPH oxidase proteins in rabbit aorta. Aortic sections were incubated with control nonimmune mouse serum ( A ) or monoclonal antibodies against p22 phox (1:100) ( B and C ), gp91 phox (1:200) ( D ), p47 phox (1:20) ( E ), and p67 phox (1:20) ( F ) at the dilutions shown, followed by gold-conjugated goat anti-mouse antibody. The sections were developed as described in Methods . Magnification is 190× ( A and C–F ) and 95× ( B ). The sections shown represent at least three stained sections for each antibody.

Article Snippet: After washing with blotto, the sections were incubated for 60 min at 25°C with 5 nm gold-conjugated goat anti-mouse antibody (Zymed) diluted 1:50 in the same buffer.

Techniques: Incubation, Staining

Journal: BMC Infectious Diseases

Article Title: Identification of circulating biomarkers in sera of Plasmodium knowlesi-infected malaria patients – comparison against Plasmodium vivax infection

doi: 10.1186/s12879-015-0786-2

Figure Lengend Snippet: Western blot of P. knowlesi pooled sera probed with P. vivax pooled sera. Unfractionated, pooled serum samples from patient is subjected to 2-DE, transferred onto nitrocellulose membranes, and probed with pooled sera (as primary antibody) followed by monoclonal anti-human IgM-HRP (as secondary antibody).

Article Snippet: After another washing step, the membranes were incubated with monoclonal anti-human Immunoglobulin M (IgM) conjugated to horseradish peroxidase (HRP) (Invitrogen, USA) at a dilution of 1:6,000 for 1 h at room temperature.

Techniques: Western Blot

Journal: BMC Infectious Diseases

Article Title: Identification of circulating biomarkers in sera of Plasmodium knowlesi-infected malaria patients – comparison against Plasmodium vivax infection

doi: 10.1186/s12879-015-0786-2

Figure Lengend Snippet: Western blot results following 2-DE. (a) normal pooled sera probed with normal pooled sera, (b) normal pooled sera probed with P. knowlesi pooled sera, (c) P. knowlesi pooled sera probed with normal pooled sera, (d) P. knowlesi pooled sera probed with P. knowlesi pooled sera, Unfractionated, pooled serum samples from patients and normal controls were subjected to 2-DE, transferred onto nitrocellulose membranes, and probed with pooled sera (as primary antibody) followed by monoclonal anti-human IgM-HRP (as secondary antibody).

Article Snippet: After another washing step, the membranes were incubated with monoclonal anti-human Immunoglobulin M (IgM) conjugated to horseradish peroxidase (HRP) (Invitrogen, USA) at a dilution of 1:6,000 for 1 h at room temperature.

Techniques: Western Blot