polyclonal anti human ifn beta antibodies  (R&D Systems)

Journal: International Journal of Molecular Sciences

Article Title: The Generation of Genetically Engineered Human Induced Pluripotent Stem Cells Overexpressing IFN-β for Future Experimental and Clinically Oriented Studies

doi: 10.3390/ijms252212456

Figure Lengend Snippet: IFNB-iPSCs express functional IFN-β. IFNB-iPSCs and K7-iPSCs were cultured in parallel and used for RNA isolation; the preparation of cell extracts; and the collection of cell culture supernatants. ( a ) IFNB-iPSCs display an increased expression of IFNB as compared with the parental K7-iPSC line. RNAs isolated from IFNB-iPSCs and K7-iPSCs were subjected to RT-PCR; IFNB expression values were normalized relative to GAPDH , and fold changes were calculated as relative IFNB mRNA levels in IFNB-iPSCs relative to K7-iPSCs (2 −∆∆Cq ). Dotted line, fold change = 2 (significance threshold). Data are shown as boxes and whiskers with minimal and maximal values (K7-iPSCs, LA8-iPSCs, and LC8-iPSCs, summarized results of at least 3 independent experiments; LE4-iPSCs, one experiment, technical replicates). Note that LA8-iPSCs and LC8-iPSCs bear a homozygous IFNB insertion, whereas LE4-iPSCs are heterozygous. The significance of the differences was determined using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. ( b , c ) IFNB-iPSCs express IFN-β at the protein level. ( b ) IFNB-iPSCs and K7-iPSCs were pelleted, frozen, and analyzed in a Western blot. The graph shows the relative densitometric values of IFN-β normalized to β-actin. ( c ) The supernatants were collected from IFNB-iPSCs and K7-iPSCs and analyzed using ELISA (the results of two independent experiments; in each of them, K7-iPSCs, LA8-iPSCs, and LC8-iPSCs were cultured in parallel and independently of cells of another experiment). ( d ) The supernatants of IFNB-iPSCs exhibit IFN-β-like functional activity. The supernatants were obtained from IFNB-iPSC and K7-iPSC cultures and were added to THP-1 macrophage-like cells pre-activated with PMA. Control THP-1 cells were cultured in the absence of iPSC supernatants. THP-1 RNA was isolated from all cultures, and the expressions of ISGs were analyzed in RT-qPCR using RPL27 as a housekeeping gene. Data are shown as boxes and whiskers with minimal and maximal values and individual points. The representative results of one out of two experiments are presented. Numbers on the graphs show the FDRs (Benjamini, Krieger, and Yekutieli correction for multiple comparisons). FDRs < 0.05 were considered as significant; for the comparison of IFNB-iPSCs and K7-iPSCs, FDRs > 0.05 are also shown. The differences between LA8-iPSCs and LC8-iPSCs were insignificant. PMA, phorbol 12-myristate-13-acetate.

Article Snippet: The membrane was blocked with a 5% non-fat dry milk (Cell Signaling Technology, Danvers, MA, USA) in a TNT buffer (10 mM of Tris-HCl, pH 7.5, 150 mM of NaCl, 0.1% Tween-20) and incubated with the following primary antibodies (4 °C, overnight, 1% milk): rabbit anti-human-IFN-β polyclonal antibodies (1:500, FNab10475, FineTest Biotech, Wuhan, Hubei, China), rabbit anti-human-PAX6 polyclonal antibodies (1:500, PAH446Ra01, Cloud-Clone Corp.), mouse anti-human-OCT-4 monoclonal antibody (1:1000, ab184665, Abcam, Cambridge, UK), mouse anti-human-actin-β antibodies (1:10,000, Abcam); and rabbit anti-human-HSP90 polyclonal antibodies (1:10,000, Sigma-Aldrich).

Techniques: Functional Assay, Cell Culture, Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Activity Assay, Control, Quantitative RT-PCR, Comparison

Journal: International Journal of Molecular Sciences

Article Title: The Generation of Genetically Engineered Human Induced Pluripotent Stem Cells Overexpressing IFN-β for Future Experimental and Clinically Oriented Studies

doi: 10.3390/ijms252212456

Figure Lengend Snippet: Exogenous IFN-β disrupts the expression of ectoderm-associated genes in differentiating parental K7-iPSCs. K7-iPSCs were subjected to spontaneous ( a ) or directed ( b – d ) differentiation in the absence or in the presence of recombinant human IFN-β; at the end of the differentiation, the expression of ectoderm-associated markers was analyzed using RT-PCR (housekeeping gene, RPL27 ) and compared with that observed in original K7-iPSCs. The significance of the differences was determined using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. ( a ) Changes in the expression of ectoderm-associated genes in spontaneously differentiated EBs. Gene expression was analyzed on day 20. Data are shown as boxes and whiskers with minimal and maximal values. The summarized results of two independent experiments are shown. ( b ) Light microscopy of K7-iPSCs differentiating in the absence or in the presence of IFN-β. Made on differentiation days 1 and 7. ( c ) Changes in the expression of ectoderm-associated genes following the directed differentiation of K7-iPSCs in the STEMdiff™ Trilineage Ectoderm Medium. Gene expression was analyzed on day 7 (as recommended by the manufacturer). Data are shown as boxes and whiskers with minimal and maximal values. The representative results of one out of two experiments are presented. ( d ) Changes in the expression of the IFNB gene following the directed differentiation of K7-iPSCs in the STEMdiff™ Trilineage Ectoderm Medium (the representative results of one out of two experiments).

Article Snippet: The membrane was blocked with a 5% non-fat dry milk (Cell Signaling Technology, Danvers, MA, USA) in a TNT buffer (10 mM of Tris-HCl, pH 7.5, 150 mM of NaCl, 0.1% Tween-20) and incubated with the following primary antibodies (4 °C, overnight, 1% milk): rabbit anti-human-IFN-β polyclonal antibodies (1:500, FNab10475, FineTest Biotech, Wuhan, Hubei, China), rabbit anti-human-PAX6 polyclonal antibodies (1:500, PAH446Ra01, Cloud-Clone Corp.), mouse anti-human-OCT-4 monoclonal antibody (1:1000, ab184665, Abcam, Cambridge, UK), mouse anti-human-actin-β antibodies (1:10,000, Abcam); and rabbit anti-human-HSP90 polyclonal antibodies (1:10,000, Sigma-Aldrich).

Techniques: Expressing, Recombinant, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Light Microscopy

Journal: Journal of Virology

Article Title: Virus Multiplicity of Infection Affects Type I Interferon Subtype Induction Profiles and Interferon-Stimulated Genes

doi: 10.1128/JVI.01727-15

Figure Lengend Snippet: U937 cells and primary monocytes produce IFN-α and IFN-β proteins upon SeV infection. U937 cells and primary monocytes were infected with SeV (150 or 1.5 HA U/ml). Supernatants were harvested at 4, 8, 16, and 24 h p.i. and assayed for IFN-α and IFN-β proteins by ELISA. (A and B) IFN-α protein levels in U937 cells and primary monocytes infected with 150 HA U/ml (A) and 1.5 HA U/ml (B). (C and D) IFN-β protein levels in U937 cells and primary monocytes infected with 150 HA U/ml (C) and 1.5 HA U/ml (D). UI, uninfected. The monocyte data are from 3 different donors. The U937 cell data are from 3 biological replicates. The error bars indicate the standard error of the mean.

Article Snippet: Rabbit anti-human IFN-β polyclonal antibody was used at a concentration of 1:10, as this concentration was 10 times higher than the concentration needed to completely neutralize the biological activity of 1,000 U/ml of purified IFN-β (R&D Biosciences, Minneapolis, MN) to ensure an appropriate molar excess of antibody.

Techniques: Infection, Enzyme-linked Immunosorbent Assay

Journal: Journal of Virology

Article Title: Virus Multiplicity of Infection Affects Type I Interferon Subtype Induction Profiles and Interferon-Stimulated Genes

doi: 10.1128/JVI.01727-15

Figure Lengend Snippet: IFN-β and IFN-α subtypes 1, 2, and 8 are induced in an NF-κB- and TBK1-dependent manner. (A) U937 cells were infected with 150 or 1.5 HA U/ml of SeV (S) for 3 h in the presence or absence of BX795 (BX), and pTBK1 and pIRF3 protein expression was measured via Western blotting. Actin was measured as a loading control. (B) Same as panel A, except cells were infected in the presence or absence of BAY11-7802 (BAY), and pIκB protein expression was measured. (C) U937 cells were infected with either 150 or 1.5 HA U/ml of SeV for 8 h in the presence or absence of the following inhibitors: BX795 (BX), BAY11-7802 (BAY), or chloroquine (Ch). (C to F) mRNA expression of IFN-α1 (C), IFN-α2 (D), IFN-α8 (E), and IFN-β (F) was measured via qRT-PCR. A one-way ANOVA with Bonferroni posttest analysis was performed. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. The data shown are from 3 biological replicates performed in duplicate. qRT-PCR data are shown as the percent relative expression of HPRT. The error bars indicate the standard error of the mean.

Article Snippet: Rabbit anti-human IFN-β polyclonal antibody was used at a concentration of 1:10, as this concentration was 10 times higher than the concentration needed to completely neutralize the biological activity of 1,000 U/ml of purified IFN-β (R&D Biosciences, Minneapolis, MN) to ensure an appropriate molar excess of antibody.

Techniques: Infection, Expressing, Western Blot, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: Virus Multiplicity of Infection Affects Type I Interferon Subtype Induction Profiles and Interferon-Stimulated Genes

doi: 10.1128/JVI.01727-15

Figure Lengend Snippet: Infection with 1.5 and 150 HA U/ml of SeV results in distinct profiles of IFN-α subtype induction. U937 cells were infected with either 1.5 or 150 HA U/ml of SeV for 4, 8, 16, 24, and 48 p.i. RNA was harvested at each time point, and IFN-α and IFN-β subtype RNA was measured via qRT-PCR. (A) IFN-β and IFN-α subtypes in cells infected with 1.5 HA U/ml. (B) IFN-β and IFN-α subtypes in cells infected with 150 HA U/ml. The data are from 3 biological replicates performed in duplicate. Statistics were performed using one-way ANOVA with Bonferroni posttest analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. qRT-PCR data are shown as the percent relative expression of HPRT. (C) U937 cells were infected with either live or inactivated SeV (150 HA U/ml) for 24 h. The supernatants were harvested and then used to pretreat fresh U937 cells prior to EMCV infection. After 72 h, cell viability was measured via MTT assay, and the ability of the supernatants to restrict EMCV-induced CPE was analyzed. The error bars indicate the standard error of the mean.

Article Snippet: Rabbit anti-human IFN-β polyclonal antibody was used at a concentration of 1:10, as this concentration was 10 times higher than the concentration needed to completely neutralize the biological activity of 1,000 U/ml of purified IFN-β (R&D Biosciences, Minneapolis, MN) to ensure an appropriate molar excess of antibody.

Techniques: Infection, Quantitative RT-PCR, Expressing, MTT Assay

Journal: Journal of Virology

Article Title: Virus Multiplicity of Infection Affects Type I Interferon Subtype Induction Profiles and Interferon-Stimulated Genes

doi: 10.1128/JVI.01727-15

Figure Lengend Snippet: Total IFN-α mRNA and protein induction is more reliant on IFNAR2 signaling in cells infected with 1.5 HA U/ml of SeV than in cells infected with 150 HA U/ml. (A) qRT-PCR analysis of PKR gene expression in cells infected with either 1.5 or 150 HA U/ml of SeV in the presence or absence of IFNAR2-neutralizing antibody. (B and C) Total IFN-α mRNA in cells infected with either 1.5 (B) or 150 (C) HA U/ml of SeV in the presence or absence of IFNAR-neutralizing antibody (NA). (D) Supernatants (sup) from cells infected with either 1.5 or 150 HA U/ml of SeV in the presence or absence of IFNAR NA (NA) for 24 h were used to pretreat fresh U937 cells in an antiviral assay using EMCV as the challenge virus. Cell viability was measured 72 h post-EMCV infection. Cell control, no pretreatment or infection; virus control, EMCV infected, no pretreatment. (E and F) ELISA measuring IFN-α protein (left) and IFN-β protein (right) in supernatants from cells infected with 1.5 (E) or 150 (F) HA U/ml of SeV in the presence or absence of IFNAR2 NA. Statistics were performed using a two-way ANOVA (B and C) and one-way ANOVA (A and D to F). Bonferroni posttest analyses were performed. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. The data are representative of 3 biological replicates performed in duplicate. qRT-PCR data are shown as the percent relative expression of HPRT. The error bars indicate the standard error of the mean.

Article Snippet: Rabbit anti-human IFN-β polyclonal antibody was used at a concentration of 1:10, as this concentration was 10 times higher than the concentration needed to completely neutralize the biological activity of 1,000 U/ml of purified IFN-β (R&D Biosciences, Minneapolis, MN) to ensure an appropriate molar excess of antibody.

Techniques: Infection, Quantitative RT-PCR, Expressing, Antiviral Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Virology

Article Title: Virus Multiplicity of Infection Affects Type I Interferon Subtype Induction Profiles and Interferon-Stimulated Genes

doi: 10.1128/JVI.01727-15

Figure Lengend Snippet: Neutralizing IFN-α and IFN-β abolishes mRNA induction of IFN-α4, -6, -7, -10, and -17. U937 cells were infected with 1.5 HA U/ml of SeV for 24 h in the presence or absence of neutralizing antibodies to total IFN-α, IFN-β, IFN-α plus IFN-β (IFNAB) or IFNAR2 (IFNAR NA). mRNA expression of IFN-α4, -6, -7, -10, and -17 was measured via qRT-PCR. One-way ANOVA with Bonferroni posttest analysis was performed. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. The data shown are from 2 biological replicates performed in duplicate. qRT-PCR data are shown as the percent relative expression of HPRT. The error bars indicate the standard error of the mean.

Article Snippet: Rabbit anti-human IFN-β polyclonal antibody was used at a concentration of 1:10, as this concentration was 10 times higher than the concentration needed to completely neutralize the biological activity of 1,000 U/ml of purified IFN-β (R&D Biosciences, Minneapolis, MN) to ensure an appropriate molar excess of antibody.

Techniques: Infection, Expressing, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: Virus Multiplicity of Infection Affects Type I Interferon Subtype Induction Profiles and Interferon-Stimulated Genes

doi: 10.1128/JVI.01727-15

Figure Lengend Snippet: IFN-β and IFN-α subtypes 1, 2 and 8 are initially induced early in an IFNAR-independent manner in both virus infections. U937 cells were infected with either 1.5 (A) or 150 (B) HA U/ml of SeV for 8 and 24 h in the presence or absence of IFNAR NA. mRNA expression of IFN-α1, -2, and -8 and IFN-β was measured via qRT-PCR. A one-way ANOVA with Bonferroni posttest analysis was performed. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001; ns, not significant. The data shown are from 3 biological replicates performed in duplicate. qRT-PCR data are shown as the percent relative expression of HPRT. The error bars indicate the standard error of the mean.

Article Snippet: Rabbit anti-human IFN-β polyclonal antibody was used at a concentration of 1:10, as this concentration was 10 times higher than the concentration needed to completely neutralize the biological activity of 1,000 U/ml of purified IFN-β (R&D Biosciences, Minneapolis, MN) to ensure an appropriate molar excess of antibody.

Techniques: Infection, Expressing, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: Virus Multiplicity of Infection Affects Type I Interferon Subtype Induction Profiles and Interferon-Stimulated Genes

doi: 10.1128/JVI.01727-15

Figure Lengend Snippet: IFN-α subtypes 5, 14, 16, and 21 are induced in an IFNAR-dependent manner in cells infected with 1.5 HA U/ml and an IFNAR-independent manner in cells infected with 150 HA U/ml. (A and C) U937 cells were infected with either 1.5 (A) or 150 (C) HA U/ml of SeV for 8 and 24 h in the presence or absence of IFNAR NA. mRNA expression of IFN-α5, -14, -16, and -21 was measured via qRT-PCR. The data shown are from 3 biological replicates performed in duplicate technical replicates. (B) U937 cells were infected with 1.5 HA U/ml of SeV for 24 h in the presence or absence of neutralizing antibodies to total IFN-α, IFN-β, IFN-α plus IFN-β (IFNAB), or IFNAR2 (IFNAR NA). mRNA expression of IFN-α5, -14, -16, and -21 was measured via qRT-PCR. The data are from 2 biological replicates performed in duplicate. A one-way ANOVA with a Bonferroni posttest analysis was performed. ****, P < 0.0001. qRT-PCR data are shown as the percent relative expression of HPRT. The error bars indicate the standard error of the mean.

Article Snippet: Rabbit anti-human IFN-β polyclonal antibody was used at a concentration of 1:10, as this concentration was 10 times higher than the concentration needed to completely neutralize the biological activity of 1,000 U/ml of purified IFN-β (R&D Biosciences, Minneapolis, MN) to ensure an appropriate molar excess of antibody.

Techniques: Infection, Expressing, Quantitative RT-PCR