anti irak m  (ProSci Incorporated)


Bioz Verified Symbol ProSci Incorporated is a verified supplier
Bioz Manufacturer Symbol ProSci Incorporated manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 90

    Structured Review

    ProSci Incorporated anti irak m
    <t>Increased</t> <t>IRAK-M</t> expression in pDCs lacking Blimp-1 contributes to impaired IFN-I production. (A,B) RT-qPCR showing Irak3 mRNA levels (A) , IRAK-M and Blimp-1 protein levels (B) in Ctrl-ER and CKO-ER FLpDCs treated with 4-OHT and then stimulated with 1 µM CpG-A. The quantitation of Blimp-1 in (B) was presented by the ratios of Blimp-1 band intensity vs. Lamin-B band intensity at each time point. The quantitation of IRAK-M in (B) was presented by the ratios of IRAK-M band intensity vs. actin band intensity at each time point. (C) Five putative Blimp-1 consensus binding sites were identified within 5 kb upstream and downstream of the Irak3 transcriptional start site (TSS, indicated by an arrow). (D) ChIP assay using chromatin isolated from FLpDCs following 4 h stimulation with 1 µM CpG-A showing the levels of binding of Blimp-1 at various putative sites. Gapdh was used as the negative control locus. (E) IFN-α production by Blimp-1-deficient and control FLpDCs transfected with control siRNA (siCtrl) or siRNA-pools with three different siRNAs against Irak3 (siIrak3) and stimulated with 1 µM CpG-A for 16 h. (F) Model of the action of Blimp-1 in the regulation of induction of IFN-I signaling in pDCs. Abbreviations: Rac, Ras-related C3 botulinum toxin substrate; IRAK-M, interleukin-1 receptor-associated kinase M; OPN, osteopontin; pDCs, plasmacytoid dendritic cells; IFN-I, type I IFN; siRNA, small-interfering RNA; Irak3, interleukin-1 receptor-associated kinase 3 ; ChIP, chromatin immunoprecipitation; RT-qPCR, RT-quantitative PCR. Data represent the mean ± SEM and were analyzed by two-tailed unpaired Student’s t -test [ n = 4 in (A) and 3 in (D,E) ]. * p < 0.05; ** p < 0.01. N.S. = no significant difference.
    Anti Irak M, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti irak m/product/ProSci Incorporated
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti irak m - by Bioz Stars, 2024-06
    90/100 stars

    Images

    1) Product Images from "Blimp-1-Mediated Pathway Promotes Type I IFN Production in Plasmacytoid Dendritic Cells by Targeting to Interleukin-1 Receptor-Associated Kinase M"

    Article Title: Blimp-1-Mediated Pathway Promotes Type I IFN Production in Plasmacytoid Dendritic Cells by Targeting to Interleukin-1 Receptor-Associated Kinase M

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01828

    Increased IRAK-M expression in pDCs lacking Blimp-1 contributes to impaired IFN-I production. (A,B) RT-qPCR showing Irak3 mRNA levels (A) , IRAK-M and Blimp-1 protein levels (B) in Ctrl-ER and CKO-ER FLpDCs treated with 4-OHT and then stimulated with 1 µM CpG-A. The quantitation of Blimp-1 in (B) was presented by the ratios of Blimp-1 band intensity vs. Lamin-B band intensity at each time point. The quantitation of IRAK-M in (B) was presented by the ratios of IRAK-M band intensity vs. actin band intensity at each time point. (C) Five putative Blimp-1 consensus binding sites were identified within 5 kb upstream and downstream of the Irak3 transcriptional start site (TSS, indicated by an arrow). (D) ChIP assay using chromatin isolated from FLpDCs following 4 h stimulation with 1 µM CpG-A showing the levels of binding of Blimp-1 at various putative sites. Gapdh was used as the negative control locus. (E) IFN-α production by Blimp-1-deficient and control FLpDCs transfected with control siRNA (siCtrl) or siRNA-pools with three different siRNAs against Irak3 (siIrak3) and stimulated with 1 µM CpG-A for 16 h. (F) Model of the action of Blimp-1 in the regulation of induction of IFN-I signaling in pDCs. Abbreviations: Rac, Ras-related C3 botulinum toxin substrate; IRAK-M, interleukin-1 receptor-associated kinase M; OPN, osteopontin; pDCs, plasmacytoid dendritic cells; IFN-I, type I IFN; siRNA, small-interfering RNA; Irak3, interleukin-1 receptor-associated kinase 3 ; ChIP, chromatin immunoprecipitation; RT-qPCR, RT-quantitative PCR. Data represent the mean ± SEM and were analyzed by two-tailed unpaired Student’s t -test [ n = 4 in (A) and 3 in (D,E) ]. * p < 0.05; ** p < 0.01. N.S. = no significant difference.
    Figure Legend Snippet: Increased IRAK-M expression in pDCs lacking Blimp-1 contributes to impaired IFN-I production. (A,B) RT-qPCR showing Irak3 mRNA levels (A) , IRAK-M and Blimp-1 protein levels (B) in Ctrl-ER and CKO-ER FLpDCs treated with 4-OHT and then stimulated with 1 µM CpG-A. The quantitation of Blimp-1 in (B) was presented by the ratios of Blimp-1 band intensity vs. Lamin-B band intensity at each time point. The quantitation of IRAK-M in (B) was presented by the ratios of IRAK-M band intensity vs. actin band intensity at each time point. (C) Five putative Blimp-1 consensus binding sites were identified within 5 kb upstream and downstream of the Irak3 transcriptional start site (TSS, indicated by an arrow). (D) ChIP assay using chromatin isolated from FLpDCs following 4 h stimulation with 1 µM CpG-A showing the levels of binding of Blimp-1 at various putative sites. Gapdh was used as the negative control locus. (E) IFN-α production by Blimp-1-deficient and control FLpDCs transfected with control siRNA (siCtrl) or siRNA-pools with three different siRNAs against Irak3 (siIrak3) and stimulated with 1 µM CpG-A for 16 h. (F) Model of the action of Blimp-1 in the regulation of induction of IFN-I signaling in pDCs. Abbreviations: Rac, Ras-related C3 botulinum toxin substrate; IRAK-M, interleukin-1 receptor-associated kinase M; OPN, osteopontin; pDCs, plasmacytoid dendritic cells; IFN-I, type I IFN; siRNA, small-interfering RNA; Irak3, interleukin-1 receptor-associated kinase 3 ; ChIP, chromatin immunoprecipitation; RT-qPCR, RT-quantitative PCR. Data represent the mean ± SEM and were analyzed by two-tailed unpaired Student’s t -test [ n = 4 in (A) and 3 in (D,E) ]. * p < 0.05; ** p < 0.01. N.S. = no significant difference.

    Techniques Used: Expressing, Quantitative RT-PCR, Quantitation Assay, Binding Assay, Isolation, Negative Control, Transfection, Small Interfering RNA, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Two Tailed Test

    anti irak m  (ProSci Incorporated)


    Bioz Verified Symbol ProSci Incorporated is a verified supplier
    Bioz Manufacturer Symbol ProSci Incorporated manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 90

    Structured Review

    ProSci Incorporated anti irak m
    <t>Increased</t> <t>IRAK-M</t> expression in pDCs lacking Blimp-1 contributes to impaired IFN-I production. (A,B) RT-qPCR showing Irak3 mRNA levels (A) , IRAK-M and Blimp-1 protein levels (B) in Ctrl-ER and CKO-ER FLpDCs treated with 4-OHT and then stimulated with 1 µM CpG-A. The quantitation of Blimp-1 in (B) was presented by the ratios of Blimp-1 band intensity vs. Lamin-B band intensity at each time point. The quantitation of IRAK-M in (B) was presented by the ratios of IRAK-M band intensity vs. actin band intensity at each time point. (C) Five putative Blimp-1 consensus binding sites were identified within 5 kb upstream and downstream of the Irak3 transcriptional start site (TSS, indicated by an arrow). (D) ChIP assay using chromatin isolated from FLpDCs following 4 h stimulation with 1 µM CpG-A showing the levels of binding of Blimp-1 at various putative sites. Gapdh was used as the negative control locus. (E) IFN-α production by Blimp-1-deficient and control FLpDCs transfected with control siRNA (siCtrl) or siRNA-pools with three different siRNAs against Irak3 (siIrak3) and stimulated with 1 µM CpG-A for 16 h. (F) Model of the action of Blimp-1 in the regulation of induction of IFN-I signaling in pDCs. Abbreviations: Rac, Ras-related C3 botulinum toxin substrate; IRAK-M, interleukin-1 receptor-associated kinase M; OPN, osteopontin; pDCs, plasmacytoid dendritic cells; IFN-I, type I IFN; siRNA, small-interfering RNA; Irak3, interleukin-1 receptor-associated kinase 3 ; ChIP, chromatin immunoprecipitation; RT-qPCR, RT-quantitative PCR. Data represent the mean ± SEM and were analyzed by two-tailed unpaired Student’s t -test [ n = 4 in (A) and 3 in (D,E) ]. * p < 0.05; ** p < 0.01. N.S. = no significant difference.
    Anti Irak M, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti irak m/product/ProSci Incorporated
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti irak m - by Bioz Stars, 2024-06
    90/100 stars

    Images

    1) Product Images from "Blimp-1-Mediated Pathway Promotes Type I IFN Production in Plasmacytoid Dendritic Cells by Targeting to Interleukin-1 Receptor-Associated Kinase M"

    Article Title: Blimp-1-Mediated Pathway Promotes Type I IFN Production in Plasmacytoid Dendritic Cells by Targeting to Interleukin-1 Receptor-Associated Kinase M

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01828

    Increased IRAK-M expression in pDCs lacking Blimp-1 contributes to impaired IFN-I production. (A,B) RT-qPCR showing Irak3 mRNA levels (A) , IRAK-M and Blimp-1 protein levels (B) in Ctrl-ER and CKO-ER FLpDCs treated with 4-OHT and then stimulated with 1 µM CpG-A. The quantitation of Blimp-1 in (B) was presented by the ratios of Blimp-1 band intensity vs. Lamin-B band intensity at each time point. The quantitation of IRAK-M in (B) was presented by the ratios of IRAK-M band intensity vs. actin band intensity at each time point. (C) Five putative Blimp-1 consensus binding sites were identified within 5 kb upstream and downstream of the Irak3 transcriptional start site (TSS, indicated by an arrow). (D) ChIP assay using chromatin isolated from FLpDCs following 4 h stimulation with 1 µM CpG-A showing the levels of binding of Blimp-1 at various putative sites. Gapdh was used as the negative control locus. (E) IFN-α production by Blimp-1-deficient and control FLpDCs transfected with control siRNA (siCtrl) or siRNA-pools with three different siRNAs against Irak3 (siIrak3) and stimulated with 1 µM CpG-A for 16 h. (F) Model of the action of Blimp-1 in the regulation of induction of IFN-I signaling in pDCs. Abbreviations: Rac, Ras-related C3 botulinum toxin substrate; IRAK-M, interleukin-1 receptor-associated kinase M; OPN, osteopontin; pDCs, plasmacytoid dendritic cells; IFN-I, type I IFN; siRNA, small-interfering RNA; Irak3, interleukin-1 receptor-associated kinase 3 ; ChIP, chromatin immunoprecipitation; RT-qPCR, RT-quantitative PCR. Data represent the mean ± SEM and were analyzed by two-tailed unpaired Student’s t -test [ n = 4 in (A) and 3 in (D,E) ]. * p < 0.05; ** p < 0.01. N.S. = no significant difference.
    Figure Legend Snippet: Increased IRAK-M expression in pDCs lacking Blimp-1 contributes to impaired IFN-I production. (A,B) RT-qPCR showing Irak3 mRNA levels (A) , IRAK-M and Blimp-1 protein levels (B) in Ctrl-ER and CKO-ER FLpDCs treated with 4-OHT and then stimulated with 1 µM CpG-A. The quantitation of Blimp-1 in (B) was presented by the ratios of Blimp-1 band intensity vs. Lamin-B band intensity at each time point. The quantitation of IRAK-M in (B) was presented by the ratios of IRAK-M band intensity vs. actin band intensity at each time point. (C) Five putative Blimp-1 consensus binding sites were identified within 5 kb upstream and downstream of the Irak3 transcriptional start site (TSS, indicated by an arrow). (D) ChIP assay using chromatin isolated from FLpDCs following 4 h stimulation with 1 µM CpG-A showing the levels of binding of Blimp-1 at various putative sites. Gapdh was used as the negative control locus. (E) IFN-α production by Blimp-1-deficient and control FLpDCs transfected with control siRNA (siCtrl) or siRNA-pools with three different siRNAs against Irak3 (siIrak3) and stimulated with 1 µM CpG-A for 16 h. (F) Model of the action of Blimp-1 in the regulation of induction of IFN-I signaling in pDCs. Abbreviations: Rac, Ras-related C3 botulinum toxin substrate; IRAK-M, interleukin-1 receptor-associated kinase M; OPN, osteopontin; pDCs, plasmacytoid dendritic cells; IFN-I, type I IFN; siRNA, small-interfering RNA; Irak3, interleukin-1 receptor-associated kinase 3 ; ChIP, chromatin immunoprecipitation; RT-qPCR, RT-quantitative PCR. Data represent the mean ± SEM and were analyzed by two-tailed unpaired Student’s t -test [ n = 4 in (A) and 3 in (D,E) ]. * p < 0.05; ** p < 0.01. N.S. = no significant difference.

    Techniques Used: Expressing, Quantitative RT-PCR, Quantitation Assay, Binding Assay, Isolation, Negative Control, Transfection, Small Interfering RNA, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Two Tailed Test

    anti irak m  (ProSci Incorporated)


    Bioz Verified Symbol ProSci Incorporated is a verified supplier
    Bioz Manufacturer Symbol ProSci Incorporated manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 90

    Structured Review

    ProSci Incorporated anti irak m
    Anti Irak M, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti irak m/product/ProSci Incorporated
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti irak m - by Bioz Stars, 2024-06
    90/100 stars

    Images

    anti irak m  (ProSci Incorporated)


    Bioz Verified Symbol ProSci Incorporated is a verified supplier
    Bioz Manufacturer Symbol ProSci Incorporated manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 90

    Structured Review

    ProSci Incorporated anti irak m
    Anti Irak M, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti irak m/product/ProSci Incorporated
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti irak m - by Bioz Stars, 2024-06
    90/100 stars

    Images

    irak m  (ProSci Incorporated)


    Bioz Verified Symbol ProSci Incorporated is a verified supplier
    Bioz Manufacturer Symbol ProSci Incorporated manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86

    Structured Review

    ProSci Incorporated irak m
    ( a ) <t>IRAK-M</t> mRNA expression in human bronchial epithelial BEAS-2B cells treated with DEX (0.01, 0.1, 1, 10, 100 or 1,000 nM) for 1 h, followed by the stimulation of NTHi for 5 h. ( b ) IRAK-M mRNA expression in human lung epithelial A549 cells stimulated with DEX (100 nM) for 1 h, followed by NTHi for indicated time. ( c ) A549 cells were treated with DEX (100 nM) for 1 h, followed by NTHi for 12 h. IRAK-M protein was detected by anti-IRAK-M antibody. ( d ) IRAK-M protein expression was quantified from three independent experiments. ( e ) IRAK-M mRNA expression in primary NHBE cells treated with DEX (10, 100 or 1,000 nM) for 1 h and subjected to NTHi stimulation for 5 h. ( f ) Human peripheral blood CD14 + monocytes were differentiated to macrophages by culturing them with granulocyte–macrophage colony-stimulating factor for 7 days. IRAK-M mRNA was determined after the macrophages were treated with DEX (100 nM) for 1 h, followed by stimulation of NTHi for 5 h. ( g ) Mice were stimulated with DEX (1 mg kg −1 , intraperitoneally) for 2 h and intratracheally inoculated with NTHi (1 × 10 7 c.f.u.) for 24 h. The mRNA expression in alveolar macrophages was assessed by qPCR. ( h ) The IRAK-M mRNA expression in lung was assessed by qPCR. ( i ) Immunostaining of mouse lung with <t>control</t> <t>IgG</t> or anti-IRAK-M antibody by LSAB (Labelled Streptavidin Biotin) staining system (Scale bar, 50 μm; magnification, × 400). ( j ) Treatment-blind observers scored the IRAK-M expression from the histology results. ( k ) The lung sections were stained by using indicated antibodies. The arrows and arrowheads indicate the epithelial cells and macrophages merged with IRAK-M expression, respectively. (Scale bar, 50 μm; magnification, × 400. Data in a , b , d - h , n =3; j , n =6) are mean±s.d. * P< 0.05; t- test.
    Irak M, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/irak m/product/ProSci Incorporated
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    irak m - by Bioz Stars, 2024-06
    86/100 stars

    Images

    1) Product Images from "Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M"

    Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

    Journal: Nature Communications

    doi: 10.1038/ncomms7062

    ( a ) IRAK-M mRNA expression in human bronchial epithelial BEAS-2B cells treated with DEX (0.01, 0.1, 1, 10, 100 or 1,000 nM) for 1 h, followed by the stimulation of NTHi for 5 h. ( b ) IRAK-M mRNA expression in human lung epithelial A549 cells stimulated with DEX (100 nM) for 1 h, followed by NTHi for indicated time. ( c ) A549 cells were treated with DEX (100 nM) for 1 h, followed by NTHi for 12 h. IRAK-M protein was detected by anti-IRAK-M antibody. ( d ) IRAK-M protein expression was quantified from three independent experiments. ( e ) IRAK-M mRNA expression in primary NHBE cells treated with DEX (10, 100 or 1,000 nM) for 1 h and subjected to NTHi stimulation for 5 h. ( f ) Human peripheral blood CD14 + monocytes were differentiated to macrophages by culturing them with granulocyte–macrophage colony-stimulating factor for 7 days. IRAK-M mRNA was determined after the macrophages were treated with DEX (100 nM) for 1 h, followed by stimulation of NTHi for 5 h. ( g ) Mice were stimulated with DEX (1 mg kg −1 , intraperitoneally) for 2 h and intratracheally inoculated with NTHi (1 × 10 7 c.f.u.) for 24 h. The mRNA expression in alveolar macrophages was assessed by qPCR. ( h ) The IRAK-M mRNA expression in lung was assessed by qPCR. ( i ) Immunostaining of mouse lung with control IgG or anti-IRAK-M antibody by LSAB (Labelled Streptavidin Biotin) staining system (Scale bar, 50 μm; magnification, × 400). ( j ) Treatment-blind observers scored the IRAK-M expression from the histology results. ( k ) The lung sections were stained by using indicated antibodies. The arrows and arrowheads indicate the epithelial cells and macrophages merged with IRAK-M expression, respectively. (Scale bar, 50 μm; magnification, × 400. Data in a , b , d - h , n =3; j , n =6) are mean±s.d. * P< 0.05; t- test.
    Figure Legend Snippet: ( a ) IRAK-M mRNA expression in human bronchial epithelial BEAS-2B cells treated with DEX (0.01, 0.1, 1, 10, 100 or 1,000 nM) for 1 h, followed by the stimulation of NTHi for 5 h. ( b ) IRAK-M mRNA expression in human lung epithelial A549 cells stimulated with DEX (100 nM) for 1 h, followed by NTHi for indicated time. ( c ) A549 cells were treated with DEX (100 nM) for 1 h, followed by NTHi for 12 h. IRAK-M protein was detected by anti-IRAK-M antibody. ( d ) IRAK-M protein expression was quantified from three independent experiments. ( e ) IRAK-M mRNA expression in primary NHBE cells treated with DEX (10, 100 or 1,000 nM) for 1 h and subjected to NTHi stimulation for 5 h. ( f ) Human peripheral blood CD14 + monocytes were differentiated to macrophages by culturing them with granulocyte–macrophage colony-stimulating factor for 7 days. IRAK-M mRNA was determined after the macrophages were treated with DEX (100 nM) for 1 h, followed by stimulation of NTHi for 5 h. ( g ) Mice were stimulated with DEX (1 mg kg −1 , intraperitoneally) for 2 h and intratracheally inoculated with NTHi (1 × 10 7 c.f.u.) for 24 h. The mRNA expression in alveolar macrophages was assessed by qPCR. ( h ) The IRAK-M mRNA expression in lung was assessed by qPCR. ( i ) Immunostaining of mouse lung with control IgG or anti-IRAK-M antibody by LSAB (Labelled Streptavidin Biotin) staining system (Scale bar, 50 μm; magnification, × 400). ( j ) Treatment-blind observers scored the IRAK-M expression from the histology results. ( k ) The lung sections were stained by using indicated antibodies. The arrows and arrowheads indicate the epithelial cells and macrophages merged with IRAK-M expression, respectively. (Scale bar, 50 μm; magnification, × 400. Data in a , b , d - h , n =3; j , n =6) are mean±s.d. * P< 0.05; t- test.

    Techniques Used: Expressing, Immunostaining, Staining

    ( a ) The proteins from BEAS-2B cells stably overexpressing IRAK-M and its control pcDNA3.1 (Mock) cells were detected by immunoblot with indicated antibodies. ( b ) The mRNA expression was detected by qPCR after the stable cells were treated with DEX (100 nM) and NTHi. ( c ) BEAS-2B cells were transfected with siRNA control or IRAK-M for 72 h. IRAK-M protein expression was detected by immunoblot with anti-IRAK-M antibodies (SC, Santa Cruz Biotechnology sc-100389; PS, ProSci #2355). ( d ) siRNA-transfected BEAS-2B cells were infected with NTHi followed by qPCR. ( e ) The mRNA level was calculated by following: (NTHi-induced mRNA at each concentration of DEX)/(NTHi-induced mRNA without DEX). Data in b , d , e , n =3) are mean±s.d. * P< 0.05; t- test.
    Figure Legend Snippet: ( a ) The proteins from BEAS-2B cells stably overexpressing IRAK-M and its control pcDNA3.1 (Mock) cells were detected by immunoblot with indicated antibodies. ( b ) The mRNA expression was detected by qPCR after the stable cells were treated with DEX (100 nM) and NTHi. ( c ) BEAS-2B cells were transfected with siRNA control or IRAK-M for 72 h. IRAK-M protein expression was detected by immunoblot with anti-IRAK-M antibodies (SC, Santa Cruz Biotechnology sc-100389; PS, ProSci #2355). ( d ) siRNA-transfected BEAS-2B cells were infected with NTHi followed by qPCR. ( e ) The mRNA level was calculated by following: (NTHi-induced mRNA at each concentration of DEX)/(NTHi-induced mRNA without DEX). Data in b , d , e , n =3) are mean±s.d. * P< 0.05; t- test.

    Techniques Used: Stable Transfection, Western Blot, Expressing, Transfection, Infection, Concentration Assay

    WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 10 7 c.f.u. per lung) for 24 h. ( a ) The mRNA expression in the mouse lung tissue was analysed by qPCR. ( b ) The protein level of proinflammatory mediators in BAL fluid was determined by enzyme-linked immunosorbent assay. ( c ) The mRNA expression in alveolar macrophages was analysed by qPCR. Data in a , c , n =3; b , n =9 are mean±s.d. * P< 0.05, NS, non-significant; t- test.
    Figure Legend Snippet: WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 10 7 c.f.u. per lung) for 24 h. ( a ) The mRNA expression in the mouse lung tissue was analysed by qPCR. ( b ) The protein level of proinflammatory mediators in BAL fluid was determined by enzyme-linked immunosorbent assay. ( c ) The mRNA expression in alveolar macrophages was analysed by qPCR. Data in a , c , n =3; b , n =9 are mean±s.d. * P< 0.05, NS, non-significant; t- test.

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay

    ( a – e ) WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 10 7 c.f.u. per lung) for 24 h. ( a ) Haematoxylin and eosin (H&E) staining of lung tissues from mice (Scale bar, 200 μm, magnification, × 100 in large frame; Scale bar 50 μm, magnification, × 400 in inserted frame). ( b ) Blinded histopathologic scoring of lung inflammation was performed on H&E-stained lung sections in a grade 0–3. ( c ) The number of PMN cells from BAL fluid was counted using a haemocytometer under the microscope. ( d ) Bacterial loads (c.f.u.) in lung homogenates. ( e ) The mRNA expression of mBD4 in lung was measured by qPCR ( f ) WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with lethal dose of NTHi (5 × 10 8 c.f.u. per lung) for 7 days. Survival rate was monitored for indicated days. P values were determined by Kaplan–Meier survival analysis with GraphPad Prism 5.0. Data in b =8; c , e , n =3; d =10, f =20 are mean±s.d. * P< 0.05, NS, non-significant; t- test.
    Figure Legend Snippet: ( a – e ) WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 10 7 c.f.u. per lung) for 24 h. ( a ) Haematoxylin and eosin (H&E) staining of lung tissues from mice (Scale bar, 200 μm, magnification, × 100 in large frame; Scale bar 50 μm, magnification, × 400 in inserted frame). ( b ) Blinded histopathologic scoring of lung inflammation was performed on H&E-stained lung sections in a grade 0–3. ( c ) The number of PMN cells from BAL fluid was counted using a haemocytometer under the microscope. ( d ) Bacterial loads (c.f.u.) in lung homogenates. ( e ) The mRNA expression of mBD4 in lung was measured by qPCR ( f ) WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with lethal dose of NTHi (5 × 10 8 c.f.u. per lung) for 7 days. Survival rate was monitored for indicated days. P values were determined by Kaplan–Meier survival analysis with GraphPad Prism 5.0. Data in b =8; c , e , n =3; d =10, f =20 are mean±s.d. * P< 0.05, NS, non-significant; t- test.

    Techniques Used: Staining, Microscopy, Expressing

    ( a ) Immunoblot shows that IRAK-M protein expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, TNF-α (10 ng ml −1 ), IL-1β (1 ng ml −1 ) or Pam3CSK4 (1 μg ml −1 ). ( b ) IRAK-M mRNA expression assessed by qPCR in A549 cells transfected with constitutive active form of IKKβ (IKKβ CA) with DEX (100 nM). ( c – f ), IRAK-M expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, IKKβ inhibitor (1 μM) ( c , d ) or RU486 (1 μM; e , f ). ( g ) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA GR. ( h ) BEAS-2B cells were treated with NTHi, DEX (100 nM) and actinomycin D, ActD (5 μg ml −1 ), followed by immunoblot. ( i ) The expression of IRAK-M mRNA in BEAS-2B cells treated with DEX (100 nM) and Compound A, CpdA (1 or 10 μM) followed by NTHi stimulation for 5 h. ( j ) Relative luciferase activity (RLA) of NF-κB is measured by luciferase assay. BEAS-2B cells transfected with NF-κB luciferase plasmid were treated with CpdA and NTHi. Data ( b , c , e , i , j , n =3) are mean±s.d. * P< 0.05, NS; t- test.
    Figure Legend Snippet: ( a ) Immunoblot shows that IRAK-M protein expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, TNF-α (10 ng ml −1 ), IL-1β (1 ng ml −1 ) or Pam3CSK4 (1 μg ml −1 ). ( b ) IRAK-M mRNA expression assessed by qPCR in A549 cells transfected with constitutive active form of IKKβ (IKKβ CA) with DEX (100 nM). ( c – f ), IRAK-M expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, IKKβ inhibitor (1 μM) ( c , d ) or RU486 (1 μM; e , f ). ( g ) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA GR. ( h ) BEAS-2B cells were treated with NTHi, DEX (100 nM) and actinomycin D, ActD (5 μg ml −1 ), followed by immunoblot. ( i ) The expression of IRAK-M mRNA in BEAS-2B cells treated with DEX (100 nM) and Compound A, CpdA (1 or 10 μM) followed by NTHi stimulation for 5 h. ( j ) Relative luciferase activity (RLA) of NF-κB is measured by luciferase assay. BEAS-2B cells transfected with NF-κB luciferase plasmid were treated with CpdA and NTHi. Data ( b , c , e , i , j , n =3) are mean±s.d. * P< 0.05, NS; t- test.

    Techniques Used: Western Blot, Expressing, Transfection, Luciferase, Activity Assay, Plasmid Preparation

    ( a – c ) Relative luciferase activity (RLA) was measured after the transfection of IRAK-M promoter in pGL3 basic vector and treated with DEX (100 nM) and NTHi in BEAS-2B cells ( a , b ). ( c ) MEF cells were transfected with IRAK-M-luc (−500 bp), followed by DEX (1 μM) and NTHi stimulation. ( d ) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA p65. ( e , f ) ChIP assay in BEAS-2B cells treated with DEX and NTHi for 1 h. IRAK-M promoter regions from −38 to +39 bp for GRE, from −231 to −99 bp for κB site were amplified by qPCR. ( g ) Re-ChIP assay (detailed in Methods). PCR products were detected in agarose gel. ( h , i ) ChIP assay in BEAS-2B cells treated with IKKβ inhibitor (1 μM), RU486 (1 μM), DEX (100 nM) and NTHi. Data ( a – c , n =3; e , f , n =2) are mean±s.d. * P<0.05 ; t- test.
    Figure Legend Snippet: ( a – c ) Relative luciferase activity (RLA) was measured after the transfection of IRAK-M promoter in pGL3 basic vector and treated with DEX (100 nM) and NTHi in BEAS-2B cells ( a , b ). ( c ) MEF cells were transfected with IRAK-M-luc (−500 bp), followed by DEX (1 μM) and NTHi stimulation. ( d ) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA p65. ( e , f ) ChIP assay in BEAS-2B cells treated with DEX and NTHi for 1 h. IRAK-M promoter regions from −38 to +39 bp for GRE, from −231 to −99 bp for κB site were amplified by qPCR. ( g ) Re-ChIP assay (detailed in Methods). PCR products were detected in agarose gel. ( h , i ) ChIP assay in BEAS-2B cells treated with IKKβ inhibitor (1 μM), RU486 (1 μM), DEX (100 nM) and NTHi. Data ( a – c , n =3; e , f , n =2) are mean±s.d. * P<0.05 ; t- test.

    Techniques Used: Luciferase, Activity Assay, Transfection, Plasmid Preparation, Western Blot, Expressing, Amplification, Agarose Gel Electrophoresis

    A schematic representation of suppression of inflammation by GCs via the upregulation of IRAK-M expression.
    Figure Legend Snippet: A schematic representation of suppression of inflammation by GCs via the upregulation of IRAK-M expression.

    Techniques Used: Expressing

    rabbit anti irak m  (ProSci Incorporated)


    Bioz Verified Symbol ProSci Incorporated is a verified supplier
    Bioz Manufacturer Symbol ProSci Incorporated manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 90

    Structured Review

    ProSci Incorporated rabbit anti irak m
    ( a <t>)</t> <t>IRAK-M</t> mRNA expression in human bronchial epithelial BEAS-2B cells treated with DEX (0.01, 0.1, 1, 10, 100 or 1,000 nM) for 1 h, followed by the stimulation of NTHi for 5 h. ( b ) IRAK-M mRNA expression in human lung epithelial A549 cells stimulated with DEX (100 nM) for 1 h, followed by NTHi for indicated time. ( c ) A549 cells were treated with DEX (100 nM) for 1 h, followed by NTHi for 12 h. IRAK-M protein was detected by anti-IRAK-M antibody. ( d ) IRAK-M protein expression was quantified from three independent experiments. ( e ) IRAK-M mRNA expression in primary NHBE cells treated with DEX (10, 100 or 1,000 nM) for 1 h and subjected to NTHi stimulation for 5 h. ( f ) Human peripheral blood CD14 + monocytes were differentiated to macrophages by culturing them with granulocyte–macrophage colony-stimulating factor for 7 days. IRAK-M mRNA was determined after the macrophages were treated with DEX (100 nM) for 1 h, followed by stimulation of NTHi for 5 h. ( g ) Mice were stimulated with DEX (1 mg kg −1 , intraperitoneally) for 2 h and intratracheally inoculated with NTHi (1 × 10 7 c.f.u.) for 24 h. The mRNA expression in alveolar macrophages was assessed by qPCR. ( h ) The IRAK-M mRNA expression in lung was assessed by qPCR. ( i ) Immunostaining of mouse lung with control IgG or anti-IRAK-M antibody by LSAB (Labelled Streptavidin Biotin) staining system (Scale bar, 50 μm; magnification, × 400). ( j ) Treatment-blind observers scored the IRAK-M expression from the histology results. ( k ) The lung sections were stained by using indicated antibodies. The arrows and arrowheads indicate the epithelial cells and macrophages merged with IRAK-M expression, respectively. (Scale bar, 50 μm; magnification, × 400. Data in a , b , d - h , n =3; j , n =6) are mean±s.d. * P< 0.05; t- test.
    Rabbit Anti Irak M, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti irak m/product/ProSci Incorporated
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti irak m - by Bioz Stars, 2024-06
    90/100 stars

    Images

    1) Product Images from "Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M"

    Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

    Journal: Nature Communications

    doi: 10.1038/ncomms7062

    ( a ) IRAK-M mRNA expression in human bronchial epithelial BEAS-2B cells treated with DEX (0.01, 0.1, 1, 10, 100 or 1,000 nM) for 1 h, followed by the stimulation of NTHi for 5 h. ( b ) IRAK-M mRNA expression in human lung epithelial A549 cells stimulated with DEX (100 nM) for 1 h, followed by NTHi for indicated time. ( c ) A549 cells were treated with DEX (100 nM) for 1 h, followed by NTHi for 12 h. IRAK-M protein was detected by anti-IRAK-M antibody. ( d ) IRAK-M protein expression was quantified from three independent experiments. ( e ) IRAK-M mRNA expression in primary NHBE cells treated with DEX (10, 100 or 1,000 nM) for 1 h and subjected to NTHi stimulation for 5 h. ( f ) Human peripheral blood CD14 + monocytes were differentiated to macrophages by culturing them with granulocyte–macrophage colony-stimulating factor for 7 days. IRAK-M mRNA was determined after the macrophages were treated with DEX (100 nM) for 1 h, followed by stimulation of NTHi for 5 h. ( g ) Mice were stimulated with DEX (1 mg kg −1 , intraperitoneally) for 2 h and intratracheally inoculated with NTHi (1 × 10 7 c.f.u.) for 24 h. The mRNA expression in alveolar macrophages was assessed by qPCR. ( h ) The IRAK-M mRNA expression in lung was assessed by qPCR. ( i ) Immunostaining of mouse lung with control IgG or anti-IRAK-M antibody by LSAB (Labelled Streptavidin Biotin) staining system (Scale bar, 50 μm; magnification, × 400). ( j ) Treatment-blind observers scored the IRAK-M expression from the histology results. ( k ) The lung sections were stained by using indicated antibodies. The arrows and arrowheads indicate the epithelial cells and macrophages merged with IRAK-M expression, respectively. (Scale bar, 50 μm; magnification, × 400. Data in a , b , d - h , n =3; j , n =6) are mean±s.d. * P< 0.05; t- test.
    Figure Legend Snippet: ( a ) IRAK-M mRNA expression in human bronchial epithelial BEAS-2B cells treated with DEX (0.01, 0.1, 1, 10, 100 or 1,000 nM) for 1 h, followed by the stimulation of NTHi for 5 h. ( b ) IRAK-M mRNA expression in human lung epithelial A549 cells stimulated with DEX (100 nM) for 1 h, followed by NTHi for indicated time. ( c ) A549 cells were treated with DEX (100 nM) for 1 h, followed by NTHi for 12 h. IRAK-M protein was detected by anti-IRAK-M antibody. ( d ) IRAK-M protein expression was quantified from three independent experiments. ( e ) IRAK-M mRNA expression in primary NHBE cells treated with DEX (10, 100 or 1,000 nM) for 1 h and subjected to NTHi stimulation for 5 h. ( f ) Human peripheral blood CD14 + monocytes were differentiated to macrophages by culturing them with granulocyte–macrophage colony-stimulating factor for 7 days. IRAK-M mRNA was determined after the macrophages were treated with DEX (100 nM) for 1 h, followed by stimulation of NTHi for 5 h. ( g ) Mice were stimulated with DEX (1 mg kg −1 , intraperitoneally) for 2 h and intratracheally inoculated with NTHi (1 × 10 7 c.f.u.) for 24 h. The mRNA expression in alveolar macrophages was assessed by qPCR. ( h ) The IRAK-M mRNA expression in lung was assessed by qPCR. ( i ) Immunostaining of mouse lung with control IgG or anti-IRAK-M antibody by LSAB (Labelled Streptavidin Biotin) staining system (Scale bar, 50 μm; magnification, × 400). ( j ) Treatment-blind observers scored the IRAK-M expression from the histology results. ( k ) The lung sections were stained by using indicated antibodies. The arrows and arrowheads indicate the epithelial cells and macrophages merged with IRAK-M expression, respectively. (Scale bar, 50 μm; magnification, × 400. Data in a , b , d - h , n =3; j , n =6) are mean±s.d. * P< 0.05; t- test.

    Techniques Used: Expressing, Immunostaining, Staining

    ( a ) The proteins from BEAS-2B cells stably overexpressing IRAK-M and its control pcDNA3.1 (Mock) cells were detected by immunoblot with indicated antibodies. ( b ) The mRNA expression was detected by qPCR after the stable cells were treated with DEX (100 nM) and NTHi. ( c ) BEAS-2B cells were transfected with siRNA control or IRAK-M for 72 h. IRAK-M protein expression was detected by immunoblot with anti-IRAK-M antibodies (SC, Santa Cruz Biotechnology sc-100389; PS, ProSci #2355). ( d ) siRNA-transfected BEAS-2B cells were infected with NTHi followed by qPCR. ( e ) The mRNA level was calculated by following: (NTHi-induced mRNA at each concentration of DEX)/(NTHi-induced mRNA without DEX). Data in b , d , e , n =3) are mean±s.d. * P< 0.05; t- test.
    Figure Legend Snippet: ( a ) The proteins from BEAS-2B cells stably overexpressing IRAK-M and its control pcDNA3.1 (Mock) cells were detected by immunoblot with indicated antibodies. ( b ) The mRNA expression was detected by qPCR after the stable cells were treated with DEX (100 nM) and NTHi. ( c ) BEAS-2B cells were transfected with siRNA control or IRAK-M for 72 h. IRAK-M protein expression was detected by immunoblot with anti-IRAK-M antibodies (SC, Santa Cruz Biotechnology sc-100389; PS, ProSci #2355). ( d ) siRNA-transfected BEAS-2B cells were infected with NTHi followed by qPCR. ( e ) The mRNA level was calculated by following: (NTHi-induced mRNA at each concentration of DEX)/(NTHi-induced mRNA without DEX). Data in b , d , e , n =3) are mean±s.d. * P< 0.05; t- test.

    Techniques Used: Stable Transfection, Western Blot, Expressing, Transfection, Infection, Concentration Assay

    WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 10 7 c.f.u. per lung) for 24 h. ( a ) The mRNA expression in the mouse lung tissue was analysed by qPCR. ( b ) The protein level of proinflammatory mediators in BAL fluid was determined by enzyme-linked immunosorbent assay. ( c ) The mRNA expression in alveolar macrophages was analysed by qPCR. Data in a , c , n =3; b , n =9 are mean±s.d. * P< 0.05, NS, non-significant; t- test.
    Figure Legend Snippet: WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 10 7 c.f.u. per lung) for 24 h. ( a ) The mRNA expression in the mouse lung tissue was analysed by qPCR. ( b ) The protein level of proinflammatory mediators in BAL fluid was determined by enzyme-linked immunosorbent assay. ( c ) The mRNA expression in alveolar macrophages was analysed by qPCR. Data in a , c , n =3; b , n =9 are mean±s.d. * P< 0.05, NS, non-significant; t- test.

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay

    ( a – e ) WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 10 7 c.f.u. per lung) for 24 h. ( a ) Haematoxylin and eosin (H&E) staining of lung tissues from mice (Scale bar, 200 μm, magnification, × 100 in large frame; Scale bar 50 μm, magnification, × 400 in inserted frame). ( b ) Blinded histopathologic scoring of lung inflammation was performed on H&E-stained lung sections in a grade 0–3. ( c ) The number of PMN cells from BAL fluid was counted using a haemocytometer under the microscope. ( d ) Bacterial loads (c.f.u.) in lung homogenates. ( e ) The mRNA expression of mBD4 in lung was measured by qPCR ( f ) WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with lethal dose of NTHi (5 × 10 8 c.f.u. per lung) for 7 days. Survival rate was monitored for indicated days. P values were determined by Kaplan–Meier survival analysis with GraphPad Prism 5.0. Data in b =8; c , e , n =3; d =10, f =20 are mean±s.d. * P< 0.05, NS, non-significant; t- test.
    Figure Legend Snippet: ( a – e ) WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 10 7 c.f.u. per lung) for 24 h. ( a ) Haematoxylin and eosin (H&E) staining of lung tissues from mice (Scale bar, 200 μm, magnification, × 100 in large frame; Scale bar 50 μm, magnification, × 400 in inserted frame). ( b ) Blinded histopathologic scoring of lung inflammation was performed on H&E-stained lung sections in a grade 0–3. ( c ) The number of PMN cells from BAL fluid was counted using a haemocytometer under the microscope. ( d ) Bacterial loads (c.f.u.) in lung homogenates. ( e ) The mRNA expression of mBD4 in lung was measured by qPCR ( f ) WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with lethal dose of NTHi (5 × 10 8 c.f.u. per lung) for 7 days. Survival rate was monitored for indicated days. P values were determined by Kaplan–Meier survival analysis with GraphPad Prism 5.0. Data in b =8; c , e , n =3; d =10, f =20 are mean±s.d. * P< 0.05, NS, non-significant; t- test.

    Techniques Used: Staining, Microscopy, Expressing

    ( a ) Immunoblot shows that IRAK-M protein expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, TNF-α (10 ng ml −1 ), IL-1β (1 ng ml −1 ) or Pam3CSK4 (1 μg ml −1 ). ( b ) IRAK-M mRNA expression assessed by qPCR in A549 cells transfected with constitutive active form of IKKβ (IKKβ CA) with DEX (100 nM). ( c – f ), IRAK-M expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, IKKβ inhibitor (1 μM) ( c , d ) or RU486 (1 μM; e , f ). ( g ) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA GR. ( h ) BEAS-2B cells were treated with NTHi, DEX (100 nM) and actinomycin D, ActD (5 μg ml −1 ), followed by immunoblot. ( i ) The expression of IRAK-M mRNA in BEAS-2B cells treated with DEX (100 nM) and Compound A, CpdA (1 or 10 μM) followed by NTHi stimulation for 5 h. ( j ) Relative luciferase activity (RLA) of NF-κB is measured by luciferase assay. BEAS-2B cells transfected with NF-κB luciferase plasmid were treated with CpdA and NTHi. Data ( b , c , e , i , j , n =3) are mean±s.d. * P< 0.05, NS; t- test.
    Figure Legend Snippet: ( a ) Immunoblot shows that IRAK-M protein expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, TNF-α (10 ng ml −1 ), IL-1β (1 ng ml −1 ) or Pam3CSK4 (1 μg ml −1 ). ( b ) IRAK-M mRNA expression assessed by qPCR in A549 cells transfected with constitutive active form of IKKβ (IKKβ CA) with DEX (100 nM). ( c – f ), IRAK-M expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, IKKβ inhibitor (1 μM) ( c , d ) or RU486 (1 μM; e , f ). ( g ) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA GR. ( h ) BEAS-2B cells were treated with NTHi, DEX (100 nM) and actinomycin D, ActD (5 μg ml −1 ), followed by immunoblot. ( i ) The expression of IRAK-M mRNA in BEAS-2B cells treated with DEX (100 nM) and Compound A, CpdA (1 or 10 μM) followed by NTHi stimulation for 5 h. ( j ) Relative luciferase activity (RLA) of NF-κB is measured by luciferase assay. BEAS-2B cells transfected with NF-κB luciferase plasmid were treated with CpdA and NTHi. Data ( b , c , e , i , j , n =3) are mean±s.d. * P< 0.05, NS; t- test.

    Techniques Used: Western Blot, Expressing, Transfection, Luciferase, Activity Assay, Plasmid Preparation

    ( a – c ) Relative luciferase activity (RLA) was measured after the transfection of IRAK-M promoter in pGL3 basic vector and treated with DEX (100 nM) and NTHi in BEAS-2B cells ( a , b ). ( c ) MEF cells were transfected with IRAK-M-luc (−500 bp), followed by DEX (1 μM) and NTHi stimulation. ( d ) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA p65. ( e , f ) ChIP assay in BEAS-2B cells treated with DEX and NTHi for 1 h. IRAK-M promoter regions from −38 to +39 bp for GRE, from −231 to −99 bp for κB site were amplified by qPCR. ( g ) Re-ChIP assay (detailed in Methods). PCR products were detected in agarose gel. ( h , i ) ChIP assay in BEAS-2B cells treated with IKKβ inhibitor (1 μM), RU486 (1 μM), DEX (100 nM) and NTHi. Data ( a – c , n =3; e , f , n =2) are mean±s.d. * P<0.05 ; t- test.
    Figure Legend Snippet: ( a – c ) Relative luciferase activity (RLA) was measured after the transfection of IRAK-M promoter in pGL3 basic vector and treated with DEX (100 nM) and NTHi in BEAS-2B cells ( a , b ). ( c ) MEF cells were transfected with IRAK-M-luc (−500 bp), followed by DEX (1 μM) and NTHi stimulation. ( d ) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA p65. ( e , f ) ChIP assay in BEAS-2B cells treated with DEX and NTHi for 1 h. IRAK-M promoter regions from −38 to +39 bp for GRE, from −231 to −99 bp for κB site were amplified by qPCR. ( g ) Re-ChIP assay (detailed in Methods). PCR products were detected in agarose gel. ( h , i ) ChIP assay in BEAS-2B cells treated with IKKβ inhibitor (1 μM), RU486 (1 μM), DEX (100 nM) and NTHi. Data ( a – c , n =3; e , f , n =2) are mean±s.d. * P<0.05 ; t- test.

    Techniques Used: Luciferase, Activity Assay, Transfection, Plasmid Preparation, Western Blot, Expressing, Amplification, Agarose Gel Electrophoresis

    A schematic representation of suppression of inflammation by GCs via the upregulation of IRAK-M expression.
    Figure Legend Snippet: A schematic representation of suppression of inflammation by GCs via the upregulation of IRAK-M expression.

    Techniques Used: Expressing

    irak-m antibody  (ProSci Incorporated)


    Bioz Verified Symbol ProSci Incorporated is a verified supplier
    Bioz Manufacturer Symbol ProSci Incorporated manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 90

    Structured Review

    ProSci Incorporated irak-m antibody
    Irak M Antibody, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/irak-m antibody/product/ProSci Incorporated
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    irak-m antibody - by Bioz Stars, 2024-06
    90/100 stars

    Images

    irak m peptide  (ProSci Incorporated)


    Bioz Verified Symbol ProSci Incorporated is a verified supplier
    Bioz Manufacturer Symbol ProSci Incorporated manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 90

    Structured Review

    ProSci Incorporated irak m peptide
    ( a <t>)</t> <t>IRAK-M</t> mRNA expression in human bronchial epithelial BEAS-2B cells treated with DEX (0.01, 0.1, 1, 10, 100 or 1,000 nM) for 1 h, followed by the stimulation of NTHi for 5 h. ( b ) IRAK-M mRNA expression in human lung epithelial A549 cells stimulated with DEX (100 nM) for 1 h, followed by NTHi for indicated time. ( c ) A549 cells were treated with DEX (100 nM) for 1 h, followed by NTHi for 12 h. IRAK-M protein was detected by anti-IRAK-M antibody. ( d ) IRAK-M protein expression was quantified from three independent experiments. ( e ) IRAK-M mRNA expression in primary NHBE cells treated with DEX (10, 100 or 1,000 nM) for 1 h and subjected to NTHi stimulation for 5 h. ( f ) Human peripheral blood CD14 + monocytes were differentiated to macrophages by culturing them with granulocyte–macrophage colony-stimulating factor for 7 days. IRAK-M mRNA was determined after the macrophages were treated with DEX (100 nM) for 1 h, followed by stimulation of NTHi for 5 h. ( g ) Mice were stimulated with DEX (1 mg kg −1 , intraperitoneally) for 2 h and intratracheally inoculated with NTHi (1 × 10 7 c.f.u.) for 24 h. The mRNA expression in alveolar macrophages was assessed by qPCR. ( h ) The IRAK-M mRNA expression in lung was assessed by qPCR. ( i ) Immunostaining of mouse lung with control IgG or anti-IRAK-M antibody by LSAB (Labelled Streptavidin Biotin) staining system (Scale bar, 50 μm; magnification, × 400). ( j ) Treatment-blind observers scored the IRAK-M expression from the histology results. ( k ) The lung sections were stained by using indicated antibodies. The arrows and arrowheads indicate the epithelial cells and macrophages merged with IRAK-M expression, respectively. (Scale bar, 50 μm; magnification, × 400. Data in a , b , d - h , n =3; j , n =6) are mean±s.d. * P< 0.05; t- test.
    Irak M Peptide, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/irak m peptide/product/ProSci Incorporated
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    irak m peptide - by Bioz Stars, 2024-06
    90/100 stars

    Images

    1) Product Images from "Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M"

    Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

    Journal: Nature Communications

    doi: 10.1038/ncomms7062

    ( a ) IRAK-M mRNA expression in human bronchial epithelial BEAS-2B cells treated with DEX (0.01, 0.1, 1, 10, 100 or 1,000 nM) for 1 h, followed by the stimulation of NTHi for 5 h. ( b ) IRAK-M mRNA expression in human lung epithelial A549 cells stimulated with DEX (100 nM) for 1 h, followed by NTHi for indicated time. ( c ) A549 cells were treated with DEX (100 nM) for 1 h, followed by NTHi for 12 h. IRAK-M protein was detected by anti-IRAK-M antibody. ( d ) IRAK-M protein expression was quantified from three independent experiments. ( e ) IRAK-M mRNA expression in primary NHBE cells treated with DEX (10, 100 or 1,000 nM) for 1 h and subjected to NTHi stimulation for 5 h. ( f ) Human peripheral blood CD14 + monocytes were differentiated to macrophages by culturing them with granulocyte–macrophage colony-stimulating factor for 7 days. IRAK-M mRNA was determined after the macrophages were treated with DEX (100 nM) for 1 h, followed by stimulation of NTHi for 5 h. ( g ) Mice were stimulated with DEX (1 mg kg −1 , intraperitoneally) for 2 h and intratracheally inoculated with NTHi (1 × 10 7 c.f.u.) for 24 h. The mRNA expression in alveolar macrophages was assessed by qPCR. ( h ) The IRAK-M mRNA expression in lung was assessed by qPCR. ( i ) Immunostaining of mouse lung with control IgG or anti-IRAK-M antibody by LSAB (Labelled Streptavidin Biotin) staining system (Scale bar, 50 μm; magnification, × 400). ( j ) Treatment-blind observers scored the IRAK-M expression from the histology results. ( k ) The lung sections were stained by using indicated antibodies. The arrows and arrowheads indicate the epithelial cells and macrophages merged with IRAK-M expression, respectively. (Scale bar, 50 μm; magnification, × 400. Data in a , b , d - h , n =3; j , n =6) are mean±s.d. * P< 0.05; t- test.
    Figure Legend Snippet: ( a ) IRAK-M mRNA expression in human bronchial epithelial BEAS-2B cells treated with DEX (0.01, 0.1, 1, 10, 100 or 1,000 nM) for 1 h, followed by the stimulation of NTHi for 5 h. ( b ) IRAK-M mRNA expression in human lung epithelial A549 cells stimulated with DEX (100 nM) for 1 h, followed by NTHi for indicated time. ( c ) A549 cells were treated with DEX (100 nM) for 1 h, followed by NTHi for 12 h. IRAK-M protein was detected by anti-IRAK-M antibody. ( d ) IRAK-M protein expression was quantified from three independent experiments. ( e ) IRAK-M mRNA expression in primary NHBE cells treated with DEX (10, 100 or 1,000 nM) for 1 h and subjected to NTHi stimulation for 5 h. ( f ) Human peripheral blood CD14 + monocytes were differentiated to macrophages by culturing them with granulocyte–macrophage colony-stimulating factor for 7 days. IRAK-M mRNA was determined after the macrophages were treated with DEX (100 nM) for 1 h, followed by stimulation of NTHi for 5 h. ( g ) Mice were stimulated with DEX (1 mg kg −1 , intraperitoneally) for 2 h and intratracheally inoculated with NTHi (1 × 10 7 c.f.u.) for 24 h. The mRNA expression in alveolar macrophages was assessed by qPCR. ( h ) The IRAK-M mRNA expression in lung was assessed by qPCR. ( i ) Immunostaining of mouse lung with control IgG or anti-IRAK-M antibody by LSAB (Labelled Streptavidin Biotin) staining system (Scale bar, 50 μm; magnification, × 400). ( j ) Treatment-blind observers scored the IRAK-M expression from the histology results. ( k ) The lung sections were stained by using indicated antibodies. The arrows and arrowheads indicate the epithelial cells and macrophages merged with IRAK-M expression, respectively. (Scale bar, 50 μm; magnification, × 400. Data in a , b , d - h , n =3; j , n =6) are mean±s.d. * P< 0.05; t- test.

    Techniques Used: Expressing, Immunostaining, Staining

    ( a ) The proteins from BEAS-2B cells stably overexpressing IRAK-M and its control pcDNA3.1 (Mock) cells were detected by immunoblot with indicated antibodies. ( b ) The mRNA expression was detected by qPCR after the stable cells were treated with DEX (100 nM) and NTHi. ( c ) BEAS-2B cells were transfected with siRNA control or IRAK-M for 72 h. IRAK-M protein expression was detected by immunoblot with anti-IRAK-M antibodies (SC, Santa Cruz Biotechnology sc-100389; PS, ProSci #2355). ( d ) siRNA-transfected BEAS-2B cells were infected with NTHi followed by qPCR. ( e ) The mRNA level was calculated by following: (NTHi-induced mRNA at each concentration of DEX)/(NTHi-induced mRNA without DEX). Data in b , d , e , n =3) are mean±s.d. * P< 0.05; t- test.
    Figure Legend Snippet: ( a ) The proteins from BEAS-2B cells stably overexpressing IRAK-M and its control pcDNA3.1 (Mock) cells were detected by immunoblot with indicated antibodies. ( b ) The mRNA expression was detected by qPCR after the stable cells were treated with DEX (100 nM) and NTHi. ( c ) BEAS-2B cells were transfected with siRNA control or IRAK-M for 72 h. IRAK-M protein expression was detected by immunoblot with anti-IRAK-M antibodies (SC, Santa Cruz Biotechnology sc-100389; PS, ProSci #2355). ( d ) siRNA-transfected BEAS-2B cells were infected with NTHi followed by qPCR. ( e ) The mRNA level was calculated by following: (NTHi-induced mRNA at each concentration of DEX)/(NTHi-induced mRNA without DEX). Data in b , d , e , n =3) are mean±s.d. * P< 0.05; t- test.

    Techniques Used: Stable Transfection, Western Blot, Expressing, Transfection, Infection, Concentration Assay

    WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 10 7 c.f.u. per lung) for 24 h. ( a ) The mRNA expression in the mouse lung tissue was analysed by qPCR. ( b ) The protein level of proinflammatory mediators in BAL fluid was determined by enzyme-linked immunosorbent assay. ( c ) The mRNA expression in alveolar macrophages was analysed by qPCR. Data in a , c , n =3; b , n =9 are mean±s.d. * P< 0.05, NS, non-significant; t- test.
    Figure Legend Snippet: WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 10 7 c.f.u. per lung) for 24 h. ( a ) The mRNA expression in the mouse lung tissue was analysed by qPCR. ( b ) The protein level of proinflammatory mediators in BAL fluid was determined by enzyme-linked immunosorbent assay. ( c ) The mRNA expression in alveolar macrophages was analysed by qPCR. Data in a , c , n =3; b , n =9 are mean±s.d. * P< 0.05, NS, non-significant; t- test.

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay

    ( a – e ) WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 10 7 c.f.u. per lung) for 24 h. ( a ) Haematoxylin and eosin (H&E) staining of lung tissues from mice (Scale bar, 200 μm, magnification, × 100 in large frame; Scale bar 50 μm, magnification, × 400 in inserted frame). ( b ) Blinded histopathologic scoring of lung inflammation was performed on H&E-stained lung sections in a grade 0–3. ( c ) The number of PMN cells from BAL fluid was counted using a haemocytometer under the microscope. ( d ) Bacterial loads (c.f.u.) in lung homogenates. ( e ) The mRNA expression of mBD4 in lung was measured by qPCR ( f ) WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with lethal dose of NTHi (5 × 10 8 c.f.u. per lung) for 7 days. Survival rate was monitored for indicated days. P values were determined by Kaplan–Meier survival analysis with GraphPad Prism 5.0. Data in b =8; c , e , n =3; d =10, f =20 are mean±s.d. * P< 0.05, NS, non-significant; t- test.
    Figure Legend Snippet: ( a – e ) WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 10 7 c.f.u. per lung) for 24 h. ( a ) Haematoxylin and eosin (H&E) staining of lung tissues from mice (Scale bar, 200 μm, magnification, × 100 in large frame; Scale bar 50 μm, magnification, × 400 in inserted frame). ( b ) Blinded histopathologic scoring of lung inflammation was performed on H&E-stained lung sections in a grade 0–3. ( c ) The number of PMN cells from BAL fluid was counted using a haemocytometer under the microscope. ( d ) Bacterial loads (c.f.u.) in lung homogenates. ( e ) The mRNA expression of mBD4 in lung was measured by qPCR ( f ) WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with lethal dose of NTHi (5 × 10 8 c.f.u. per lung) for 7 days. Survival rate was monitored for indicated days. P values were determined by Kaplan–Meier survival analysis with GraphPad Prism 5.0. Data in b =8; c , e , n =3; d =10, f =20 are mean±s.d. * P< 0.05, NS, non-significant; t- test.

    Techniques Used: Staining, Microscopy, Expressing

    ( a ) Immunoblot shows that IRAK-M protein expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, TNF-α (10 ng ml −1 ), IL-1β (1 ng ml −1 ) or Pam3CSK4 (1 μg ml −1 ). ( b ) IRAK-M mRNA expression assessed by qPCR in A549 cells transfected with constitutive active form of IKKβ (IKKβ CA) with DEX (100 nM). ( c – f ), IRAK-M expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, IKKβ inhibitor (1 μM) ( c , d ) or RU486 (1 μM; e , f ). ( g ) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA GR. ( h ) BEAS-2B cells were treated with NTHi, DEX (100 nM) and actinomycin D, ActD (5 μg ml −1 ), followed by immunoblot. ( i ) The expression of IRAK-M mRNA in BEAS-2B cells treated with DEX (100 nM) and Compound A, CpdA (1 or 10 μM) followed by NTHi stimulation for 5 h. ( j ) Relative luciferase activity (RLA) of NF-κB is measured by luciferase assay. BEAS-2B cells transfected with NF-κB luciferase plasmid were treated with CpdA and NTHi. Data ( b , c , e , i , j , n =3) are mean±s.d. * P< 0.05, NS; t- test.
    Figure Legend Snippet: ( a ) Immunoblot shows that IRAK-M protein expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, TNF-α (10 ng ml −1 ), IL-1β (1 ng ml −1 ) or Pam3CSK4 (1 μg ml −1 ). ( b ) IRAK-M mRNA expression assessed by qPCR in A549 cells transfected with constitutive active form of IKKβ (IKKβ CA) with DEX (100 nM). ( c – f ), IRAK-M expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, IKKβ inhibitor (1 μM) ( c , d ) or RU486 (1 μM; e , f ). ( g ) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA GR. ( h ) BEAS-2B cells were treated with NTHi, DEX (100 nM) and actinomycin D, ActD (5 μg ml −1 ), followed by immunoblot. ( i ) The expression of IRAK-M mRNA in BEAS-2B cells treated with DEX (100 nM) and Compound A, CpdA (1 or 10 μM) followed by NTHi stimulation for 5 h. ( j ) Relative luciferase activity (RLA) of NF-κB is measured by luciferase assay. BEAS-2B cells transfected with NF-κB luciferase plasmid were treated with CpdA and NTHi. Data ( b , c , e , i , j , n =3) are mean±s.d. * P< 0.05, NS; t- test.

    Techniques Used: Western Blot, Expressing, Transfection, Luciferase, Activity Assay, Plasmid Preparation

    ( a – c ) Relative luciferase activity (RLA) was measured after the transfection of IRAK-M promoter in pGL3 basic vector and treated with DEX (100 nM) and NTHi in BEAS-2B cells ( a , b ). ( c ) MEF cells were transfected with IRAK-M-luc (−500 bp), followed by DEX (1 μM) and NTHi stimulation. ( d ) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA p65. ( e , f ) ChIP assay in BEAS-2B cells treated with DEX and NTHi for 1 h. IRAK-M promoter regions from −38 to +39 bp for GRE, from −231 to −99 bp for κB site were amplified by qPCR. ( g ) Re-ChIP assay (detailed in Methods). PCR products were detected in agarose gel. ( h , i ) ChIP assay in BEAS-2B cells treated with IKKβ inhibitor (1 μM), RU486 (1 μM), DEX (100 nM) and NTHi. Data ( a – c , n =3; e , f , n =2) are mean±s.d. * P<0.05 ; t- test.
    Figure Legend Snippet: ( a – c ) Relative luciferase activity (RLA) was measured after the transfection of IRAK-M promoter in pGL3 basic vector and treated with DEX (100 nM) and NTHi in BEAS-2B cells ( a , b ). ( c ) MEF cells were transfected with IRAK-M-luc (−500 bp), followed by DEX (1 μM) and NTHi stimulation. ( d ) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA p65. ( e , f ) ChIP assay in BEAS-2B cells treated with DEX and NTHi for 1 h. IRAK-M promoter regions from −38 to +39 bp for GRE, from −231 to −99 bp for κB site were amplified by qPCR. ( g ) Re-ChIP assay (detailed in Methods). PCR products were detected in agarose gel. ( h , i ) ChIP assay in BEAS-2B cells treated with IKKβ inhibitor (1 μM), RU486 (1 μM), DEX (100 nM) and NTHi. Data ( a – c , n =3; e , f , n =2) are mean±s.d. * P<0.05 ; t- test.

    Techniques Used: Luciferase, Activity Assay, Transfection, Plasmid Preparation, Western Blot, Expressing, Amplification, Agarose Gel Electrophoresis

    A schematic representation of suppression of inflammation by GCs via the upregulation of IRAK-M expression.
    Figure Legend Snippet: A schematic representation of suppression of inflammation by GCs via the upregulation of IRAK-M expression.

    Techniques Used: Expressing

    irak m protein  (ProSci Incorporated)


    Bioz Verified Symbol ProSci Incorporated is a verified supplier
    Bioz Manufacturer Symbol ProSci Incorporated manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 90

    Structured Review

    ProSci Incorporated irak m protein
    ( a <t>)</t> <t>IRAK-M</t> mRNA expression in human bronchial epithelial BEAS-2B cells treated with DEX (0.01, 0.1, 1, 10, 100 or 1,000 nM) for 1 h, followed by the stimulation of NTHi for 5 h. ( b ) IRAK-M mRNA expression in human lung epithelial A549 cells stimulated with DEX (100 nM) for 1 h, followed by NTHi for indicated time. ( c ) A549 cells were treated with DEX (100 nM) for 1 h, followed by NTHi for 12 h. IRAK-M protein was detected by anti-IRAK-M antibody. ( d ) IRAK-M protein expression was quantified from three independent experiments. ( e ) IRAK-M mRNA expression in primary NHBE cells treated with DEX (10, 100 or 1,000 nM) for 1 h and subjected to NTHi stimulation for 5 h. ( f ) Human peripheral blood CD14 + monocytes were differentiated to macrophages by culturing them with granulocyte–macrophage colony-stimulating factor for 7 days. IRAK-M mRNA was determined after the macrophages were treated with DEX (100 nM) for 1 h, followed by stimulation of NTHi for 5 h. ( g ) Mice were stimulated with DEX (1 mg kg −1 , intraperitoneally) for 2 h and intratracheally inoculated with NTHi (1 × 10 7 c.f.u.) for 24 h. The mRNA expression in alveolar macrophages was assessed by qPCR. ( h ) The IRAK-M mRNA expression in lung was assessed by qPCR. ( i ) Immunostaining of mouse lung with control IgG or anti-IRAK-M antibody by LSAB (Labelled Streptavidin Biotin) staining system (Scale bar, 50 μm; magnification, × 400). ( j ) Treatment-blind observers scored the IRAK-M expression from the histology results. ( k ) The lung sections were stained by using indicated antibodies. The arrows and arrowheads indicate the epithelial cells and macrophages merged with IRAK-M expression, respectively. (Scale bar, 50 μm; magnification, × 400. Data in a , b , d - h , n =3; j , n =6) are mean±s.d. * P< 0.05; t- test.
    Irak M Protein, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/irak m protein/product/ProSci Incorporated
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    irak m protein - by Bioz Stars, 2024-06
    90/100 stars

    Images

    1) Product Images from "Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M"

    Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

    Journal: Nature Communications

    doi: 10.1038/ncomms7062

    ( a ) IRAK-M mRNA expression in human bronchial epithelial BEAS-2B cells treated with DEX (0.01, 0.1, 1, 10, 100 or 1,000 nM) for 1 h, followed by the stimulation of NTHi for 5 h. ( b ) IRAK-M mRNA expression in human lung epithelial A549 cells stimulated with DEX (100 nM) for 1 h, followed by NTHi for indicated time. ( c ) A549 cells were treated with DEX (100 nM) for 1 h, followed by NTHi for 12 h. IRAK-M protein was detected by anti-IRAK-M antibody. ( d ) IRAK-M protein expression was quantified from three independent experiments. ( e ) IRAK-M mRNA expression in primary NHBE cells treated with DEX (10, 100 or 1,000 nM) for 1 h and subjected to NTHi stimulation for 5 h. ( f ) Human peripheral blood CD14 + monocytes were differentiated to macrophages by culturing them with granulocyte–macrophage colony-stimulating factor for 7 days. IRAK-M mRNA was determined after the macrophages were treated with DEX (100 nM) for 1 h, followed by stimulation of NTHi for 5 h. ( g ) Mice were stimulated with DEX (1 mg kg −1 , intraperitoneally) for 2 h and intratracheally inoculated with NTHi (1 × 10 7 c.f.u.) for 24 h. The mRNA expression in alveolar macrophages was assessed by qPCR. ( h ) The IRAK-M mRNA expression in lung was assessed by qPCR. ( i ) Immunostaining of mouse lung with control IgG or anti-IRAK-M antibody by LSAB (Labelled Streptavidin Biotin) staining system (Scale bar, 50 μm; magnification, × 400). ( j ) Treatment-blind observers scored the IRAK-M expression from the histology results. ( k ) The lung sections were stained by using indicated antibodies. The arrows and arrowheads indicate the epithelial cells and macrophages merged with IRAK-M expression, respectively. (Scale bar, 50 μm; magnification, × 400. Data in a , b , d - h , n =3; j , n =6) are mean±s.d. * P< 0.05; t- test.
    Figure Legend Snippet: ( a ) IRAK-M mRNA expression in human bronchial epithelial BEAS-2B cells treated with DEX (0.01, 0.1, 1, 10, 100 or 1,000 nM) for 1 h, followed by the stimulation of NTHi for 5 h. ( b ) IRAK-M mRNA expression in human lung epithelial A549 cells stimulated with DEX (100 nM) for 1 h, followed by NTHi for indicated time. ( c ) A549 cells were treated with DEX (100 nM) for 1 h, followed by NTHi for 12 h. IRAK-M protein was detected by anti-IRAK-M antibody. ( d ) IRAK-M protein expression was quantified from three independent experiments. ( e ) IRAK-M mRNA expression in primary NHBE cells treated with DEX (10, 100 or 1,000 nM) for 1 h and subjected to NTHi stimulation for 5 h. ( f ) Human peripheral blood CD14 + monocytes were differentiated to macrophages by culturing them with granulocyte–macrophage colony-stimulating factor for 7 days. IRAK-M mRNA was determined after the macrophages were treated with DEX (100 nM) for 1 h, followed by stimulation of NTHi for 5 h. ( g ) Mice were stimulated with DEX (1 mg kg −1 , intraperitoneally) for 2 h and intratracheally inoculated with NTHi (1 × 10 7 c.f.u.) for 24 h. The mRNA expression in alveolar macrophages was assessed by qPCR. ( h ) The IRAK-M mRNA expression in lung was assessed by qPCR. ( i ) Immunostaining of mouse lung with control IgG or anti-IRAK-M antibody by LSAB (Labelled Streptavidin Biotin) staining system (Scale bar, 50 μm; magnification, × 400). ( j ) Treatment-blind observers scored the IRAK-M expression from the histology results. ( k ) The lung sections were stained by using indicated antibodies. The arrows and arrowheads indicate the epithelial cells and macrophages merged with IRAK-M expression, respectively. (Scale bar, 50 μm; magnification, × 400. Data in a , b , d - h , n =3; j , n =6) are mean±s.d. * P< 0.05; t- test.

    Techniques Used: Expressing, Immunostaining, Staining

    ( a ) The proteins from BEAS-2B cells stably overexpressing IRAK-M and its control pcDNA3.1 (Mock) cells were detected by immunoblot with indicated antibodies. ( b ) The mRNA expression was detected by qPCR after the stable cells were treated with DEX (100 nM) and NTHi. ( c ) BEAS-2B cells were transfected with siRNA control or IRAK-M for 72 h. IRAK-M protein expression was detected by immunoblot with anti-IRAK-M antibodies (SC, Santa Cruz Biotechnology sc-100389; PS, ProSci #2355). ( d ) siRNA-transfected BEAS-2B cells were infected with NTHi followed by qPCR. ( e ) The mRNA level was calculated by following: (NTHi-induced mRNA at each concentration of DEX)/(NTHi-induced mRNA without DEX). Data in b , d , e , n =3) are mean±s.d. * P< 0.05; t- test.
    Figure Legend Snippet: ( a ) The proteins from BEAS-2B cells stably overexpressing IRAK-M and its control pcDNA3.1 (Mock) cells were detected by immunoblot with indicated antibodies. ( b ) The mRNA expression was detected by qPCR after the stable cells were treated with DEX (100 nM) and NTHi. ( c ) BEAS-2B cells were transfected with siRNA control or IRAK-M for 72 h. IRAK-M protein expression was detected by immunoblot with anti-IRAK-M antibodies (SC, Santa Cruz Biotechnology sc-100389; PS, ProSci #2355). ( d ) siRNA-transfected BEAS-2B cells were infected with NTHi followed by qPCR. ( e ) The mRNA level was calculated by following: (NTHi-induced mRNA at each concentration of DEX)/(NTHi-induced mRNA without DEX). Data in b , d , e , n =3) are mean±s.d. * P< 0.05; t- test.

    Techniques Used: Stable Transfection, Western Blot, Expressing, Transfection, Infection, Concentration Assay

    WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 10 7 c.f.u. per lung) for 24 h. ( a ) The mRNA expression in the mouse lung tissue was analysed by qPCR. ( b ) The protein level of proinflammatory mediators in BAL fluid was determined by enzyme-linked immunosorbent assay. ( c ) The mRNA expression in alveolar macrophages was analysed by qPCR. Data in a , c , n =3; b , n =9 are mean±s.d. * P< 0.05, NS, non-significant; t- test.
    Figure Legend Snippet: WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 10 7 c.f.u. per lung) for 24 h. ( a ) The mRNA expression in the mouse lung tissue was analysed by qPCR. ( b ) The protein level of proinflammatory mediators in BAL fluid was determined by enzyme-linked immunosorbent assay. ( c ) The mRNA expression in alveolar macrophages was analysed by qPCR. Data in a , c , n =3; b , n =9 are mean±s.d. * P< 0.05, NS, non-significant; t- test.

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay

    ( a – e ) WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 10 7 c.f.u. per lung) for 24 h. ( a ) Haematoxylin and eosin (H&E) staining of lung tissues from mice (Scale bar, 200 μm, magnification, × 100 in large frame; Scale bar 50 μm, magnification, × 400 in inserted frame). ( b ) Blinded histopathologic scoring of lung inflammation was performed on H&E-stained lung sections in a grade 0–3. ( c ) The number of PMN cells from BAL fluid was counted using a haemocytometer under the microscope. ( d ) Bacterial loads (c.f.u.) in lung homogenates. ( e ) The mRNA expression of mBD4 in lung was measured by qPCR ( f ) WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with lethal dose of NTHi (5 × 10 8 c.f.u. per lung) for 7 days. Survival rate was monitored for indicated days. P values were determined by Kaplan–Meier survival analysis with GraphPad Prism 5.0. Data in b =8; c , e , n =3; d =10, f =20 are mean±s.d. * P< 0.05, NS, non-significant; t- test.
    Figure Legend Snippet: ( a – e ) WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 10 7 c.f.u. per lung) for 24 h. ( a ) Haematoxylin and eosin (H&E) staining of lung tissues from mice (Scale bar, 200 μm, magnification, × 100 in large frame; Scale bar 50 μm, magnification, × 400 in inserted frame). ( b ) Blinded histopathologic scoring of lung inflammation was performed on H&E-stained lung sections in a grade 0–3. ( c ) The number of PMN cells from BAL fluid was counted using a haemocytometer under the microscope. ( d ) Bacterial loads (c.f.u.) in lung homogenates. ( e ) The mRNA expression of mBD4 in lung was measured by qPCR ( f ) WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with lethal dose of NTHi (5 × 10 8 c.f.u. per lung) for 7 days. Survival rate was monitored for indicated days. P values were determined by Kaplan–Meier survival analysis with GraphPad Prism 5.0. Data in b =8; c , e , n =3; d =10, f =20 are mean±s.d. * P< 0.05, NS, non-significant; t- test.

    Techniques Used: Staining, Microscopy, Expressing

    ( a ) Immunoblot shows that IRAK-M protein expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, TNF-α (10 ng ml −1 ), IL-1β (1 ng ml −1 ) or Pam3CSK4 (1 μg ml −1 ). ( b ) IRAK-M mRNA expression assessed by qPCR in A549 cells transfected with constitutive active form of IKKβ (IKKβ CA) with DEX (100 nM). ( c – f ), IRAK-M expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, IKKβ inhibitor (1 μM) ( c , d ) or RU486 (1 μM; e , f ). ( g ) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA GR. ( h ) BEAS-2B cells were treated with NTHi, DEX (100 nM) and actinomycin D, ActD (5 μg ml −1 ), followed by immunoblot. ( i ) The expression of IRAK-M mRNA in BEAS-2B cells treated with DEX (100 nM) and Compound A, CpdA (1 or 10 μM) followed by NTHi stimulation for 5 h. ( j ) Relative luciferase activity (RLA) of NF-κB is measured by luciferase assay. BEAS-2B cells transfected with NF-κB luciferase plasmid were treated with CpdA and NTHi. Data ( b , c , e , i , j , n =3) are mean±s.d. * P< 0.05, NS; t- test.
    Figure Legend Snippet: ( a ) Immunoblot shows that IRAK-M protein expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, TNF-α (10 ng ml −1 ), IL-1β (1 ng ml −1 ) or Pam3CSK4 (1 μg ml −1 ). ( b ) IRAK-M mRNA expression assessed by qPCR in A549 cells transfected with constitutive active form of IKKβ (IKKβ CA) with DEX (100 nM). ( c – f ), IRAK-M expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, IKKβ inhibitor (1 μM) ( c , d ) or RU486 (1 μM; e , f ). ( g ) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA GR. ( h ) BEAS-2B cells were treated with NTHi, DEX (100 nM) and actinomycin D, ActD (5 μg ml −1 ), followed by immunoblot. ( i ) The expression of IRAK-M mRNA in BEAS-2B cells treated with DEX (100 nM) and Compound A, CpdA (1 or 10 μM) followed by NTHi stimulation for 5 h. ( j ) Relative luciferase activity (RLA) of NF-κB is measured by luciferase assay. BEAS-2B cells transfected with NF-κB luciferase plasmid were treated with CpdA and NTHi. Data ( b , c , e , i , j , n =3) are mean±s.d. * P< 0.05, NS; t- test.

    Techniques Used: Western Blot, Expressing, Transfection, Luciferase, Activity Assay, Plasmid Preparation

    ( a – c ) Relative luciferase activity (RLA) was measured after the transfection of IRAK-M promoter in pGL3 basic vector and treated with DEX (100 nM) and NTHi in BEAS-2B cells ( a , b ). ( c ) MEF cells were transfected with IRAK-M-luc (−500 bp), followed by DEX (1 μM) and NTHi stimulation. ( d ) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA p65. ( e , f ) ChIP assay in BEAS-2B cells treated with DEX and NTHi for 1 h. IRAK-M promoter regions from −38 to +39 bp for GRE, from −231 to −99 bp for κB site were amplified by qPCR. ( g ) Re-ChIP assay (detailed in Methods). PCR products were detected in agarose gel. ( h , i ) ChIP assay in BEAS-2B cells treated with IKKβ inhibitor (1 μM), RU486 (1 μM), DEX (100 nM) and NTHi. Data ( a – c , n =3; e , f , n =2) are mean±s.d. * P<0.05 ; t- test.
    Figure Legend Snippet: ( a – c ) Relative luciferase activity (RLA) was measured after the transfection of IRAK-M promoter in pGL3 basic vector and treated with DEX (100 nM) and NTHi in BEAS-2B cells ( a , b ). ( c ) MEF cells were transfected with IRAK-M-luc (−500 bp), followed by DEX (1 μM) and NTHi stimulation. ( d ) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA p65. ( e , f ) ChIP assay in BEAS-2B cells treated with DEX and NTHi for 1 h. IRAK-M promoter regions from −38 to +39 bp for GRE, from −231 to −99 bp for κB site were amplified by qPCR. ( g ) Re-ChIP assay (detailed in Methods). PCR products were detected in agarose gel. ( h , i ) ChIP assay in BEAS-2B cells treated with IKKβ inhibitor (1 μM), RU486 (1 μM), DEX (100 nM) and NTHi. Data ( a – c , n =3; e , f , n =2) are mean±s.d. * P<0.05 ; t- test.

    Techniques Used: Luciferase, Activity Assay, Transfection, Plasmid Preparation, Western Blot, Expressing, Amplification, Agarose Gel Electrophoresis

    A schematic representation of suppression of inflammation by GCs via the upregulation of IRAK-M expression.
    Figure Legend Snippet: A schematic representation of suppression of inflammation by GCs via the upregulation of IRAK-M expression.

    Techniques Used: Expressing

    irak m  (ProSci Incorporated)


    Bioz Verified Symbol ProSci Incorporated is a verified supplier
    Bioz Manufacturer Symbol ProSci Incorporated manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 90

    Structured Review

    ProSci Incorporated irak m
    Irak M, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/irak m/product/ProSci Incorporated
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    irak m - by Bioz Stars, 2024-06
    90/100 stars

    Images

    irak m  (ProSci Incorporated)


    Bioz Verified Symbol ProSci Incorporated is a verified supplier
    Bioz Manufacturer Symbol ProSci Incorporated manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 90

    Structured Review

    ProSci Incorporated irak m
    Irak M, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/irak m/product/ProSci Incorporated
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    irak m - by Bioz Stars, 2024-06
    90/100 stars

    Images

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 90
    ProSci Incorporated anti irak m
    <t>Increased</t> <t>IRAK-M</t> expression in pDCs lacking Blimp-1 contributes to impaired IFN-I production. (A,B) RT-qPCR showing Irak3 mRNA levels (A) , IRAK-M and Blimp-1 protein levels (B) in Ctrl-ER and CKO-ER FLpDCs treated with 4-OHT and then stimulated with 1 µM CpG-A. The quantitation of Blimp-1 in (B) was presented by the ratios of Blimp-1 band intensity vs. Lamin-B band intensity at each time point. The quantitation of IRAK-M in (B) was presented by the ratios of IRAK-M band intensity vs. actin band intensity at each time point. (C) Five putative Blimp-1 consensus binding sites were identified within 5 kb upstream and downstream of the Irak3 transcriptional start site (TSS, indicated by an arrow). (D) ChIP assay using chromatin isolated from FLpDCs following 4 h stimulation with 1 µM CpG-A showing the levels of binding of Blimp-1 at various putative sites. Gapdh was used as the negative control locus. (E) IFN-α production by Blimp-1-deficient and control FLpDCs transfected with control siRNA (siCtrl) or siRNA-pools with three different siRNAs against Irak3 (siIrak3) and stimulated with 1 µM CpG-A for 16 h. (F) Model of the action of Blimp-1 in the regulation of induction of IFN-I signaling in pDCs. Abbreviations: Rac, Ras-related C3 botulinum toxin substrate; IRAK-M, interleukin-1 receptor-associated kinase M; OPN, osteopontin; pDCs, plasmacytoid dendritic cells; IFN-I, type I IFN; siRNA, small-interfering RNA; Irak3, interleukin-1 receptor-associated kinase 3 ; ChIP, chromatin immunoprecipitation; RT-qPCR, RT-quantitative PCR. Data represent the mean ± SEM and were analyzed by two-tailed unpaired Student’s t -test [ n = 4 in (A) and 3 in (D,E) ]. * p < 0.05; ** p < 0.01. N.S. = no significant difference.
    Anti Irak M, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti irak m/product/ProSci Incorporated
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti irak m - by Bioz Stars, 2024-06
    90/100 stars
      Buy from Supplier

    86
    ProSci Incorporated irak m
    ( a ) <t>IRAK-M</t> mRNA expression in human bronchial epithelial BEAS-2B cells treated with DEX (0.01, 0.1, 1, 10, 100 or 1,000 nM) for 1 h, followed by the stimulation of NTHi for 5 h. ( b ) IRAK-M mRNA expression in human lung epithelial A549 cells stimulated with DEX (100 nM) for 1 h, followed by NTHi for indicated time. ( c ) A549 cells were treated with DEX (100 nM) for 1 h, followed by NTHi for 12 h. IRAK-M protein was detected by anti-IRAK-M antibody. ( d ) IRAK-M protein expression was quantified from three independent experiments. ( e ) IRAK-M mRNA expression in primary NHBE cells treated with DEX (10, 100 or 1,000 nM) for 1 h and subjected to NTHi stimulation for 5 h. ( f ) Human peripheral blood CD14 + monocytes were differentiated to macrophages by culturing them with granulocyte–macrophage colony-stimulating factor for 7 days. IRAK-M mRNA was determined after the macrophages were treated with DEX (100 nM) for 1 h, followed by stimulation of NTHi for 5 h. ( g ) Mice were stimulated with DEX (1 mg kg −1 , intraperitoneally) for 2 h and intratracheally inoculated with NTHi (1 × 10 7 c.f.u.) for 24 h. The mRNA expression in alveolar macrophages was assessed by qPCR. ( h ) The IRAK-M mRNA expression in lung was assessed by qPCR. ( i ) Immunostaining of mouse lung with <t>control</t> <t>IgG</t> or anti-IRAK-M antibody by LSAB (Labelled Streptavidin Biotin) staining system (Scale bar, 50 μm; magnification, × 400). ( j ) Treatment-blind observers scored the IRAK-M expression from the histology results. ( k ) The lung sections were stained by using indicated antibodies. The arrows and arrowheads indicate the epithelial cells and macrophages merged with IRAK-M expression, respectively. (Scale bar, 50 μm; magnification, × 400. Data in a , b , d - h , n =3; j , n =6) are mean±s.d. * P< 0.05; t- test.
    Irak M, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/irak m/product/ProSci Incorporated
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    irak m - by Bioz Stars, 2024-06
    86/100 stars
      Buy from Supplier

    90
    ProSci Incorporated rabbit anti irak m
    ( a <t>)</t> <t>IRAK-M</t> mRNA expression in human bronchial epithelial BEAS-2B cells treated with DEX (0.01, 0.1, 1, 10, 100 or 1,000 nM) for 1 h, followed by the stimulation of NTHi for 5 h. ( b ) IRAK-M mRNA expression in human lung epithelial A549 cells stimulated with DEX (100 nM) for 1 h, followed by NTHi for indicated time. ( c ) A549 cells were treated with DEX (100 nM) for 1 h, followed by NTHi for 12 h. IRAK-M protein was detected by anti-IRAK-M antibody. ( d ) IRAK-M protein expression was quantified from three independent experiments. ( e ) IRAK-M mRNA expression in primary NHBE cells treated with DEX (10, 100 or 1,000 nM) for 1 h and subjected to NTHi stimulation for 5 h. ( f ) Human peripheral blood CD14 + monocytes were differentiated to macrophages by culturing them with granulocyte–macrophage colony-stimulating factor for 7 days. IRAK-M mRNA was determined after the macrophages were treated with DEX (100 nM) for 1 h, followed by stimulation of NTHi for 5 h. ( g ) Mice were stimulated with DEX (1 mg kg −1 , intraperitoneally) for 2 h and intratracheally inoculated with NTHi (1 × 10 7 c.f.u.) for 24 h. The mRNA expression in alveolar macrophages was assessed by qPCR. ( h ) The IRAK-M mRNA expression in lung was assessed by qPCR. ( i ) Immunostaining of mouse lung with control IgG or anti-IRAK-M antibody by LSAB (Labelled Streptavidin Biotin) staining system (Scale bar, 50 μm; magnification, × 400). ( j ) Treatment-blind observers scored the IRAK-M expression from the histology results. ( k ) The lung sections were stained by using indicated antibodies. The arrows and arrowheads indicate the epithelial cells and macrophages merged with IRAK-M expression, respectively. (Scale bar, 50 μm; magnification, × 400. Data in a , b , d - h , n =3; j , n =6) are mean±s.d. * P< 0.05; t- test.
    Rabbit Anti Irak M, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti irak m/product/ProSci Incorporated
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti irak m - by Bioz Stars, 2024-06
    90/100 stars
      Buy from Supplier

    90
    ProSci Incorporated irak-m antibody
    ( a <t>)</t> <t>IRAK-M</t> mRNA expression in human bronchial epithelial BEAS-2B cells treated with DEX (0.01, 0.1, 1, 10, 100 or 1,000 nM) for 1 h, followed by the stimulation of NTHi for 5 h. ( b ) IRAK-M mRNA expression in human lung epithelial A549 cells stimulated with DEX (100 nM) for 1 h, followed by NTHi for indicated time. ( c ) A549 cells were treated with DEX (100 nM) for 1 h, followed by NTHi for 12 h. IRAK-M protein was detected by anti-IRAK-M antibody. ( d ) IRAK-M protein expression was quantified from three independent experiments. ( e ) IRAK-M mRNA expression in primary NHBE cells treated with DEX (10, 100 or 1,000 nM) for 1 h and subjected to NTHi stimulation for 5 h. ( f ) Human peripheral blood CD14 + monocytes were differentiated to macrophages by culturing them with granulocyte–macrophage colony-stimulating factor for 7 days. IRAK-M mRNA was determined after the macrophages were treated with DEX (100 nM) for 1 h, followed by stimulation of NTHi for 5 h. ( g ) Mice were stimulated with DEX (1 mg kg −1 , intraperitoneally) for 2 h and intratracheally inoculated with NTHi (1 × 10 7 c.f.u.) for 24 h. The mRNA expression in alveolar macrophages was assessed by qPCR. ( h ) The IRAK-M mRNA expression in lung was assessed by qPCR. ( i ) Immunostaining of mouse lung with control IgG or anti-IRAK-M antibody by LSAB (Labelled Streptavidin Biotin) staining system (Scale bar, 50 μm; magnification, × 400). ( j ) Treatment-blind observers scored the IRAK-M expression from the histology results. ( k ) The lung sections were stained by using indicated antibodies. The arrows and arrowheads indicate the epithelial cells and macrophages merged with IRAK-M expression, respectively. (Scale bar, 50 μm; magnification, × 400. Data in a , b , d - h , n =3; j , n =6) are mean±s.d. * P< 0.05; t- test.
    Irak M Antibody, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/irak-m antibody/product/ProSci Incorporated
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    irak-m antibody - by Bioz Stars, 2024-06
    90/100 stars
      Buy from Supplier

    90
    ProSci Incorporated irak m peptide
    ( a <t>)</t> <t>IRAK-M</t> mRNA expression in human bronchial epithelial BEAS-2B cells treated with DEX (0.01, 0.1, 1, 10, 100 or 1,000 nM) for 1 h, followed by the stimulation of NTHi for 5 h. ( b ) IRAK-M mRNA expression in human lung epithelial A549 cells stimulated with DEX (100 nM) for 1 h, followed by NTHi for indicated time. ( c ) A549 cells were treated with DEX (100 nM) for 1 h, followed by NTHi for 12 h. IRAK-M protein was detected by anti-IRAK-M antibody. ( d ) IRAK-M protein expression was quantified from three independent experiments. ( e ) IRAK-M mRNA expression in primary NHBE cells treated with DEX (10, 100 or 1,000 nM) for 1 h and subjected to NTHi stimulation for 5 h. ( f ) Human peripheral blood CD14 + monocytes were differentiated to macrophages by culturing them with granulocyte–macrophage colony-stimulating factor for 7 days. IRAK-M mRNA was determined after the macrophages were treated with DEX (100 nM) for 1 h, followed by stimulation of NTHi for 5 h. ( g ) Mice were stimulated with DEX (1 mg kg −1 , intraperitoneally) for 2 h and intratracheally inoculated with NTHi (1 × 10 7 c.f.u.) for 24 h. The mRNA expression in alveolar macrophages was assessed by qPCR. ( h ) The IRAK-M mRNA expression in lung was assessed by qPCR. ( i ) Immunostaining of mouse lung with control IgG or anti-IRAK-M antibody by LSAB (Labelled Streptavidin Biotin) staining system (Scale bar, 50 μm; magnification, × 400). ( j ) Treatment-blind observers scored the IRAK-M expression from the histology results. ( k ) The lung sections were stained by using indicated antibodies. The arrows and arrowheads indicate the epithelial cells and macrophages merged with IRAK-M expression, respectively. (Scale bar, 50 μm; magnification, × 400. Data in a , b , d - h , n =3; j , n =6) are mean±s.d. * P< 0.05; t- test.
    Irak M Peptide, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/irak m peptide/product/ProSci Incorporated
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    irak m peptide - by Bioz Stars, 2024-06
    90/100 stars
      Buy from Supplier

    90
    ProSci Incorporated irak m protein
    ( a <t>)</t> <t>IRAK-M</t> mRNA expression in human bronchial epithelial BEAS-2B cells treated with DEX (0.01, 0.1, 1, 10, 100 or 1,000 nM) for 1 h, followed by the stimulation of NTHi for 5 h. ( b ) IRAK-M mRNA expression in human lung epithelial A549 cells stimulated with DEX (100 nM) for 1 h, followed by NTHi for indicated time. ( c ) A549 cells were treated with DEX (100 nM) for 1 h, followed by NTHi for 12 h. IRAK-M protein was detected by anti-IRAK-M antibody. ( d ) IRAK-M protein expression was quantified from three independent experiments. ( e ) IRAK-M mRNA expression in primary NHBE cells treated with DEX (10, 100 or 1,000 nM) for 1 h and subjected to NTHi stimulation for 5 h. ( f ) Human peripheral blood CD14 + monocytes were differentiated to macrophages by culturing them with granulocyte–macrophage colony-stimulating factor for 7 days. IRAK-M mRNA was determined after the macrophages were treated with DEX (100 nM) for 1 h, followed by stimulation of NTHi for 5 h. ( g ) Mice were stimulated with DEX (1 mg kg −1 , intraperitoneally) for 2 h and intratracheally inoculated with NTHi (1 × 10 7 c.f.u.) for 24 h. The mRNA expression in alveolar macrophages was assessed by qPCR. ( h ) The IRAK-M mRNA expression in lung was assessed by qPCR. ( i ) Immunostaining of mouse lung with control IgG or anti-IRAK-M antibody by LSAB (Labelled Streptavidin Biotin) staining system (Scale bar, 50 μm; magnification, × 400). ( j ) Treatment-blind observers scored the IRAK-M expression from the histology results. ( k ) The lung sections were stained by using indicated antibodies. The arrows and arrowheads indicate the epithelial cells and macrophages merged with IRAK-M expression, respectively. (Scale bar, 50 μm; magnification, × 400. Data in a , b , d - h , n =3; j , n =6) are mean±s.d. * P< 0.05; t- test.
    Irak M Protein, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/irak m protein/product/ProSci Incorporated
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    irak m protein - by Bioz Stars, 2024-06
    90/100 stars
      Buy from Supplier

    Image Search Results


    Increased IRAK-M expression in pDCs lacking Blimp-1 contributes to impaired IFN-I production. (A,B) RT-qPCR showing Irak3 mRNA levels (A) , IRAK-M and Blimp-1 protein levels (B) in Ctrl-ER and CKO-ER FLpDCs treated with 4-OHT and then stimulated with 1 µM CpG-A. The quantitation of Blimp-1 in (B) was presented by the ratios of Blimp-1 band intensity vs. Lamin-B band intensity at each time point. The quantitation of IRAK-M in (B) was presented by the ratios of IRAK-M band intensity vs. actin band intensity at each time point. (C) Five putative Blimp-1 consensus binding sites were identified within 5 kb upstream and downstream of the Irak3 transcriptional start site (TSS, indicated by an arrow). (D) ChIP assay using chromatin isolated from FLpDCs following 4 h stimulation with 1 µM CpG-A showing the levels of binding of Blimp-1 at various putative sites. Gapdh was used as the negative control locus. (E) IFN-α production by Blimp-1-deficient and control FLpDCs transfected with control siRNA (siCtrl) or siRNA-pools with three different siRNAs against Irak3 (siIrak3) and stimulated with 1 µM CpG-A for 16 h. (F) Model of the action of Blimp-1 in the regulation of induction of IFN-I signaling in pDCs. Abbreviations: Rac, Ras-related C3 botulinum toxin substrate; IRAK-M, interleukin-1 receptor-associated kinase M; OPN, osteopontin; pDCs, plasmacytoid dendritic cells; IFN-I, type I IFN; siRNA, small-interfering RNA; Irak3, interleukin-1 receptor-associated kinase 3 ; ChIP, chromatin immunoprecipitation; RT-qPCR, RT-quantitative PCR. Data represent the mean ± SEM and were analyzed by two-tailed unpaired Student’s t -test [ n = 4 in (A) and 3 in (D,E) ]. * p < 0.05; ** p < 0.01. N.S. = no significant difference.

    Journal: Frontiers in Immunology

    Article Title: Blimp-1-Mediated Pathway Promotes Type I IFN Production in Plasmacytoid Dendritic Cells by Targeting to Interleukin-1 Receptor-Associated Kinase M

    doi: 10.3389/fimmu.2018.01828

    Figure Lengend Snippet: Increased IRAK-M expression in pDCs lacking Blimp-1 contributes to impaired IFN-I production. (A,B) RT-qPCR showing Irak3 mRNA levels (A) , IRAK-M and Blimp-1 protein levels (B) in Ctrl-ER and CKO-ER FLpDCs treated with 4-OHT and then stimulated with 1 µM CpG-A. The quantitation of Blimp-1 in (B) was presented by the ratios of Blimp-1 band intensity vs. Lamin-B band intensity at each time point. The quantitation of IRAK-M in (B) was presented by the ratios of IRAK-M band intensity vs. actin band intensity at each time point. (C) Five putative Blimp-1 consensus binding sites were identified within 5 kb upstream and downstream of the Irak3 transcriptional start site (TSS, indicated by an arrow). (D) ChIP assay using chromatin isolated from FLpDCs following 4 h stimulation with 1 µM CpG-A showing the levels of binding of Blimp-1 at various putative sites. Gapdh was used as the negative control locus. (E) IFN-α production by Blimp-1-deficient and control FLpDCs transfected with control siRNA (siCtrl) or siRNA-pools with three different siRNAs against Irak3 (siIrak3) and stimulated with 1 µM CpG-A for 16 h. (F) Model of the action of Blimp-1 in the regulation of induction of IFN-I signaling in pDCs. Abbreviations: Rac, Ras-related C3 botulinum toxin substrate; IRAK-M, interleukin-1 receptor-associated kinase M; OPN, osteopontin; pDCs, plasmacytoid dendritic cells; IFN-I, type I IFN; siRNA, small-interfering RNA; Irak3, interleukin-1 receptor-associated kinase 3 ; ChIP, chromatin immunoprecipitation; RT-qPCR, RT-quantitative PCR. Data represent the mean ± SEM and were analyzed by two-tailed unpaired Student’s t -test [ n = 4 in (A) and 3 in (D,E) ]. * p < 0.05; ** p < 0.01. N.S. = no significant difference.

    Article Snippet: The blots were probed with anti-IRF7 antibody (EPR4718; abcam), anti-Lamin-B (M-20; Santa Cruz Biotechnology), anti-IKKα (Cell Signaling), anti-AKT (Cell Signaling), anti-Osteopontin (Abcam), anti-p65 (C-20; Santa Cruz Biotechnology), anti-P50 (Santa Cruz Biotechnology), anti-STAT1 (Cell Signaling), anti-IRAK-M (ProSci), and anti-Blimp-1 (Abcam).

    Techniques: Expressing, Quantitative RT-PCR, Quantitation Assay, Binding Assay, Isolation, Negative Control, Transfection, Small Interfering RNA, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Two Tailed Test

    ( a ) IRAK-M mRNA expression in human bronchial epithelial BEAS-2B cells treated with DEX (0.01, 0.1, 1, 10, 100 or 1,000 nM) for 1 h, followed by the stimulation of NTHi for 5 h. ( b ) IRAK-M mRNA expression in human lung epithelial A549 cells stimulated with DEX (100 nM) for 1 h, followed by NTHi for indicated time. ( c ) A549 cells were treated with DEX (100 nM) for 1 h, followed by NTHi for 12 h. IRAK-M protein was detected by anti-IRAK-M antibody. ( d ) IRAK-M protein expression was quantified from three independent experiments. ( e ) IRAK-M mRNA expression in primary NHBE cells treated with DEX (10, 100 or 1,000 nM) for 1 h and subjected to NTHi stimulation for 5 h. ( f ) Human peripheral blood CD14 + monocytes were differentiated to macrophages by culturing them with granulocyte–macrophage colony-stimulating factor for 7 days. IRAK-M mRNA was determined after the macrophages were treated with DEX (100 nM) for 1 h, followed by stimulation of NTHi for 5 h. ( g ) Mice were stimulated with DEX (1 mg kg −1 , intraperitoneally) for 2 h and intratracheally inoculated with NTHi (1 × 10 7 c.f.u.) for 24 h. The mRNA expression in alveolar macrophages was assessed by qPCR. ( h ) The IRAK-M mRNA expression in lung was assessed by qPCR. ( i ) Immunostaining of mouse lung with control IgG or anti-IRAK-M antibody by LSAB (Labelled Streptavidin Biotin) staining system (Scale bar, 50 μm; magnification, × 400). ( j ) Treatment-blind observers scored the IRAK-M expression from the histology results. ( k ) The lung sections were stained by using indicated antibodies. The arrows and arrowheads indicate the epithelial cells and macrophages merged with IRAK-M expression, respectively. (Scale bar, 50 μm; magnification, × 400. Data in a , b , d - h , n =3; j , n =6) are mean±s.d. * P< 0.05; t- test.

    Journal: Nature Communications

    Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

    doi: 10.1038/ncomms7062

    Figure Lengend Snippet: ( a ) IRAK-M mRNA expression in human bronchial epithelial BEAS-2B cells treated with DEX (0.01, 0.1, 1, 10, 100 or 1,000 nM) for 1 h, followed by the stimulation of NTHi for 5 h. ( b ) IRAK-M mRNA expression in human lung epithelial A549 cells stimulated with DEX (100 nM) for 1 h, followed by NTHi for indicated time. ( c ) A549 cells were treated with DEX (100 nM) for 1 h, followed by NTHi for 12 h. IRAK-M protein was detected by anti-IRAK-M antibody. ( d ) IRAK-M protein expression was quantified from three independent experiments. ( e ) IRAK-M mRNA expression in primary NHBE cells treated with DEX (10, 100 or 1,000 nM) for 1 h and subjected to NTHi stimulation for 5 h. ( f ) Human peripheral blood CD14 + monocytes were differentiated to macrophages by culturing them with granulocyte–macrophage colony-stimulating factor for 7 days. IRAK-M mRNA was determined after the macrophages were treated with DEX (100 nM) for 1 h, followed by stimulation of NTHi for 5 h. ( g ) Mice were stimulated with DEX (1 mg kg −1 , intraperitoneally) for 2 h and intratracheally inoculated with NTHi (1 × 10 7 c.f.u.) for 24 h. The mRNA expression in alveolar macrophages was assessed by qPCR. ( h ) The IRAK-M mRNA expression in lung was assessed by qPCR. ( i ) Immunostaining of mouse lung with control IgG or anti-IRAK-M antibody by LSAB (Labelled Streptavidin Biotin) staining system (Scale bar, 50 μm; magnification, × 400). ( j ) Treatment-blind observers scored the IRAK-M expression from the histology results. ( k ) The lung sections were stained by using indicated antibodies. The arrows and arrowheads indicate the epithelial cells and macrophages merged with IRAK-M expression, respectively. (Scale bar, 50 μm; magnification, × 400. Data in a , b , d - h , n =3; j , n =6) are mean±s.d. * P< 0.05; t- test.

    Article Snippet: Antibodies: GR (sc-8992), p65 (sc-8008, sc-372, sc-109), IRAK-M (sc-100389), α-Tubulin (sc-69969), β-actin (sc-8432), F4/80 (sc-26642), E-cadherin (sc-31020), Donkey anti-rabbit IgG-FITC (sc-2090) and bovine anti-goat IgG-TR (sc-2786) were purchased from Santa Cruz Biotechnology, IRAK-M (#2355) from ProSci, anti-rabbit HRP-linked antibody (#7074) and anti-mouse HRP-linked antibody (#7076) were from Cell Signaling. siRNAs; GR (cat# L-003424-00-0005, ON-TARGET plus Human NR3C1 (2908)-SMARTpool, p65 (cat# L-003533-00-0005, ON-TARGET plus Human RELA (5970) - SMARTpool) and control siRNA (cat# D-001810-10-05) were from Thermo Scientific Dharmacon, IRAK-M (IRAK3 (ID 11213) cat# SR307690 and control siRNA (cat# SR30004) were from OriGene.

    Techniques: Expressing, Immunostaining, Staining

    ( a ) The proteins from BEAS-2B cells stably overexpressing IRAK-M and its control pcDNA3.1 (Mock) cells were detected by immunoblot with indicated antibodies. ( b ) The mRNA expression was detected by qPCR after the stable cells were treated with DEX (100 nM) and NTHi. ( c ) BEAS-2B cells were transfected with siRNA control or IRAK-M for 72 h. IRAK-M protein expression was detected by immunoblot with anti-IRAK-M antibodies (SC, Santa Cruz Biotechnology sc-100389; PS, ProSci #2355). ( d ) siRNA-transfected BEAS-2B cells were infected with NTHi followed by qPCR. ( e ) The mRNA level was calculated by following: (NTHi-induced mRNA at each concentration of DEX)/(NTHi-induced mRNA without DEX). Data in b , d , e , n =3) are mean±s.d. * P< 0.05; t- test.

    Journal: Nature Communications

    Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

    doi: 10.1038/ncomms7062

    Figure Lengend Snippet: ( a ) The proteins from BEAS-2B cells stably overexpressing IRAK-M and its control pcDNA3.1 (Mock) cells were detected by immunoblot with indicated antibodies. ( b ) The mRNA expression was detected by qPCR after the stable cells were treated with DEX (100 nM) and NTHi. ( c ) BEAS-2B cells were transfected with siRNA control or IRAK-M for 72 h. IRAK-M protein expression was detected by immunoblot with anti-IRAK-M antibodies (SC, Santa Cruz Biotechnology sc-100389; PS, ProSci #2355). ( d ) siRNA-transfected BEAS-2B cells were infected with NTHi followed by qPCR. ( e ) The mRNA level was calculated by following: (NTHi-induced mRNA at each concentration of DEX)/(NTHi-induced mRNA without DEX). Data in b , d , e , n =3) are mean±s.d. * P< 0.05; t- test.

    Article Snippet: Antibodies: GR (sc-8992), p65 (sc-8008, sc-372, sc-109), IRAK-M (sc-100389), α-Tubulin (sc-69969), β-actin (sc-8432), F4/80 (sc-26642), E-cadherin (sc-31020), Donkey anti-rabbit IgG-FITC (sc-2090) and bovine anti-goat IgG-TR (sc-2786) were purchased from Santa Cruz Biotechnology, IRAK-M (#2355) from ProSci, anti-rabbit HRP-linked antibody (#7074) and anti-mouse HRP-linked antibody (#7076) were from Cell Signaling. siRNAs; GR (cat# L-003424-00-0005, ON-TARGET plus Human NR3C1 (2908)-SMARTpool, p65 (cat# L-003533-00-0005, ON-TARGET plus Human RELA (5970) - SMARTpool) and control siRNA (cat# D-001810-10-05) were from Thermo Scientific Dharmacon, IRAK-M (IRAK3 (ID 11213) cat# SR307690 and control siRNA (cat# SR30004) were from OriGene.

    Techniques: Stable Transfection, Western Blot, Expressing, Transfection, Infection, Concentration Assay

    WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 10 7 c.f.u. per lung) for 24 h. ( a ) The mRNA expression in the mouse lung tissue was analysed by qPCR. ( b ) The protein level of proinflammatory mediators in BAL fluid was determined by enzyme-linked immunosorbent assay. ( c ) The mRNA expression in alveolar macrophages was analysed by qPCR. Data in a , c , n =3; b , n =9 are mean±s.d. * P< 0.05, NS, non-significant; t- test.

    Journal: Nature Communications

    Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

    doi: 10.1038/ncomms7062

    Figure Lengend Snippet: WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 10 7 c.f.u. per lung) for 24 h. ( a ) The mRNA expression in the mouse lung tissue was analysed by qPCR. ( b ) The protein level of proinflammatory mediators in BAL fluid was determined by enzyme-linked immunosorbent assay. ( c ) The mRNA expression in alveolar macrophages was analysed by qPCR. Data in a , c , n =3; b , n =9 are mean±s.d. * P< 0.05, NS, non-significant; t- test.

    Article Snippet: Antibodies: GR (sc-8992), p65 (sc-8008, sc-372, sc-109), IRAK-M (sc-100389), α-Tubulin (sc-69969), β-actin (sc-8432), F4/80 (sc-26642), E-cadherin (sc-31020), Donkey anti-rabbit IgG-FITC (sc-2090) and bovine anti-goat IgG-TR (sc-2786) were purchased from Santa Cruz Biotechnology, IRAK-M (#2355) from ProSci, anti-rabbit HRP-linked antibody (#7074) and anti-mouse HRP-linked antibody (#7076) were from Cell Signaling. siRNAs; GR (cat# L-003424-00-0005, ON-TARGET plus Human NR3C1 (2908)-SMARTpool, p65 (cat# L-003533-00-0005, ON-TARGET plus Human RELA (5970) - SMARTpool) and control siRNA (cat# D-001810-10-05) were from Thermo Scientific Dharmacon, IRAK-M (IRAK3 (ID 11213) cat# SR307690 and control siRNA (cat# SR30004) were from OriGene.

    Techniques: Expressing, Enzyme-linked Immunosorbent Assay

    ( a – e ) WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 10 7 c.f.u. per lung) for 24 h. ( a ) Haematoxylin and eosin (H&E) staining of lung tissues from mice (Scale bar, 200 μm, magnification, × 100 in large frame; Scale bar 50 μm, magnification, × 400 in inserted frame). ( b ) Blinded histopathologic scoring of lung inflammation was performed on H&E-stained lung sections in a grade 0–3. ( c ) The number of PMN cells from BAL fluid was counted using a haemocytometer under the microscope. ( d ) Bacterial loads (c.f.u.) in lung homogenates. ( e ) The mRNA expression of mBD4 in lung was measured by qPCR ( f ) WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with lethal dose of NTHi (5 × 10 8 c.f.u. per lung) for 7 days. Survival rate was monitored for indicated days. P values were determined by Kaplan–Meier survival analysis with GraphPad Prism 5.0. Data in b =8; c , e , n =3; d =10, f =20 are mean±s.d. * P< 0.05, NS, non-significant; t- test.

    Journal: Nature Communications

    Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

    doi: 10.1038/ncomms7062

    Figure Lengend Snippet: ( a – e ) WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 10 7 c.f.u. per lung) for 24 h. ( a ) Haematoxylin and eosin (H&E) staining of lung tissues from mice (Scale bar, 200 μm, magnification, × 100 in large frame; Scale bar 50 μm, magnification, × 400 in inserted frame). ( b ) Blinded histopathologic scoring of lung inflammation was performed on H&E-stained lung sections in a grade 0–3. ( c ) The number of PMN cells from BAL fluid was counted using a haemocytometer under the microscope. ( d ) Bacterial loads (c.f.u.) in lung homogenates. ( e ) The mRNA expression of mBD4 in lung was measured by qPCR ( f ) WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with lethal dose of NTHi (5 × 10 8 c.f.u. per lung) for 7 days. Survival rate was monitored for indicated days. P values were determined by Kaplan–Meier survival analysis with GraphPad Prism 5.0. Data in b =8; c , e , n =3; d =10, f =20 are mean±s.d. * P< 0.05, NS, non-significant; t- test.

    Article Snippet: Antibodies: GR (sc-8992), p65 (sc-8008, sc-372, sc-109), IRAK-M (sc-100389), α-Tubulin (sc-69969), β-actin (sc-8432), F4/80 (sc-26642), E-cadherin (sc-31020), Donkey anti-rabbit IgG-FITC (sc-2090) and bovine anti-goat IgG-TR (sc-2786) were purchased from Santa Cruz Biotechnology, IRAK-M (#2355) from ProSci, anti-rabbit HRP-linked antibody (#7074) and anti-mouse HRP-linked antibody (#7076) were from Cell Signaling. siRNAs; GR (cat# L-003424-00-0005, ON-TARGET plus Human NR3C1 (2908)-SMARTpool, p65 (cat# L-003533-00-0005, ON-TARGET plus Human RELA (5970) - SMARTpool) and control siRNA (cat# D-001810-10-05) were from Thermo Scientific Dharmacon, IRAK-M (IRAK3 (ID 11213) cat# SR307690 and control siRNA (cat# SR30004) were from OriGene.

    Techniques: Staining, Microscopy, Expressing

    ( a ) Immunoblot shows that IRAK-M protein expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, TNF-α (10 ng ml −1 ), IL-1β (1 ng ml −1 ) or Pam3CSK4 (1 μg ml −1 ). ( b ) IRAK-M mRNA expression assessed by qPCR in A549 cells transfected with constitutive active form of IKKβ (IKKβ CA) with DEX (100 nM). ( c – f ), IRAK-M expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, IKKβ inhibitor (1 μM) ( c , d ) or RU486 (1 μM; e , f ). ( g ) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA GR. ( h ) BEAS-2B cells were treated with NTHi, DEX (100 nM) and actinomycin D, ActD (5 μg ml −1 ), followed by immunoblot. ( i ) The expression of IRAK-M mRNA in BEAS-2B cells treated with DEX (100 nM) and Compound A, CpdA (1 or 10 μM) followed by NTHi stimulation for 5 h. ( j ) Relative luciferase activity (RLA) of NF-κB is measured by luciferase assay. BEAS-2B cells transfected with NF-κB luciferase plasmid were treated with CpdA and NTHi. Data ( b , c , e , i , j , n =3) are mean±s.d. * P< 0.05, NS; t- test.

    Journal: Nature Communications

    Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

    doi: 10.1038/ncomms7062

    Figure Lengend Snippet: ( a ) Immunoblot shows that IRAK-M protein expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, TNF-α (10 ng ml −1 ), IL-1β (1 ng ml −1 ) or Pam3CSK4 (1 μg ml −1 ). ( b ) IRAK-M mRNA expression assessed by qPCR in A549 cells transfected with constitutive active form of IKKβ (IKKβ CA) with DEX (100 nM). ( c – f ), IRAK-M expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, IKKβ inhibitor (1 μM) ( c , d ) or RU486 (1 μM; e , f ). ( g ) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA GR. ( h ) BEAS-2B cells were treated with NTHi, DEX (100 nM) and actinomycin D, ActD (5 μg ml −1 ), followed by immunoblot. ( i ) The expression of IRAK-M mRNA in BEAS-2B cells treated with DEX (100 nM) and Compound A, CpdA (1 or 10 μM) followed by NTHi stimulation for 5 h. ( j ) Relative luciferase activity (RLA) of NF-κB is measured by luciferase assay. BEAS-2B cells transfected with NF-κB luciferase plasmid were treated with CpdA and NTHi. Data ( b , c , e , i , j , n =3) are mean±s.d. * P< 0.05, NS; t- test.

    Article Snippet: Antibodies: GR (sc-8992), p65 (sc-8008, sc-372, sc-109), IRAK-M (sc-100389), α-Tubulin (sc-69969), β-actin (sc-8432), F4/80 (sc-26642), E-cadherin (sc-31020), Donkey anti-rabbit IgG-FITC (sc-2090) and bovine anti-goat IgG-TR (sc-2786) were purchased from Santa Cruz Biotechnology, IRAK-M (#2355) from ProSci, anti-rabbit HRP-linked antibody (#7074) and anti-mouse HRP-linked antibody (#7076) were from Cell Signaling. siRNAs; GR (cat# L-003424-00-0005, ON-TARGET plus Human NR3C1 (2908)-SMARTpool, p65 (cat# L-003533-00-0005, ON-TARGET plus Human RELA (5970) - SMARTpool) and control siRNA (cat# D-001810-10-05) were from Thermo Scientific Dharmacon, IRAK-M (IRAK3 (ID 11213) cat# SR307690 and control siRNA (cat# SR30004) were from OriGene.

    Techniques: Western Blot, Expressing, Transfection, Luciferase, Activity Assay, Plasmid Preparation

    ( a – c ) Relative luciferase activity (RLA) was measured after the transfection of IRAK-M promoter in pGL3 basic vector and treated with DEX (100 nM) and NTHi in BEAS-2B cells ( a , b ). ( c ) MEF cells were transfected with IRAK-M-luc (−500 bp), followed by DEX (1 μM) and NTHi stimulation. ( d ) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA p65. ( e , f ) ChIP assay in BEAS-2B cells treated with DEX and NTHi for 1 h. IRAK-M promoter regions from −38 to +39 bp for GRE, from −231 to −99 bp for κB site were amplified by qPCR. ( g ) Re-ChIP assay (detailed in Methods). PCR products were detected in agarose gel. ( h , i ) ChIP assay in BEAS-2B cells treated with IKKβ inhibitor (1 μM), RU486 (1 μM), DEX (100 nM) and NTHi. Data ( a – c , n =3; e , f , n =2) are mean±s.d. * P<0.05 ; t- test.

    Journal: Nature Communications

    Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

    doi: 10.1038/ncomms7062

    Figure Lengend Snippet: ( a – c ) Relative luciferase activity (RLA) was measured after the transfection of IRAK-M promoter in pGL3 basic vector and treated with DEX (100 nM) and NTHi in BEAS-2B cells ( a , b ). ( c ) MEF cells were transfected with IRAK-M-luc (−500 bp), followed by DEX (1 μM) and NTHi stimulation. ( d ) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA p65. ( e , f ) ChIP assay in BEAS-2B cells treated with DEX and NTHi for 1 h. IRAK-M promoter regions from −38 to +39 bp for GRE, from −231 to −99 bp for κB site were amplified by qPCR. ( g ) Re-ChIP assay (detailed in Methods). PCR products were detected in agarose gel. ( h , i ) ChIP assay in BEAS-2B cells treated with IKKβ inhibitor (1 μM), RU486 (1 μM), DEX (100 nM) and NTHi. Data ( a – c , n =3; e , f , n =2) are mean±s.d. * P<0.05 ; t- test.

    Article Snippet: Antibodies: GR (sc-8992), p65 (sc-8008, sc-372, sc-109), IRAK-M (sc-100389), α-Tubulin (sc-69969), β-actin (sc-8432), F4/80 (sc-26642), E-cadherin (sc-31020), Donkey anti-rabbit IgG-FITC (sc-2090) and bovine anti-goat IgG-TR (sc-2786) were purchased from Santa Cruz Biotechnology, IRAK-M (#2355) from ProSci, anti-rabbit HRP-linked antibody (#7074) and anti-mouse HRP-linked antibody (#7076) were from Cell Signaling. siRNAs; GR (cat# L-003424-00-0005, ON-TARGET plus Human NR3C1 (2908)-SMARTpool, p65 (cat# L-003533-00-0005, ON-TARGET plus Human RELA (5970) - SMARTpool) and control siRNA (cat# D-001810-10-05) were from Thermo Scientific Dharmacon, IRAK-M (IRAK3 (ID 11213) cat# SR307690 and control siRNA (cat# SR30004) were from OriGene.

    Techniques: Luciferase, Activity Assay, Transfection, Plasmid Preparation, Western Blot, Expressing, Amplification, Agarose Gel Electrophoresis

    A schematic representation of suppression of inflammation by GCs via the upregulation of IRAK-M expression.

    Journal: Nature Communications

    Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

    doi: 10.1038/ncomms7062

    Figure Lengend Snippet: A schematic representation of suppression of inflammation by GCs via the upregulation of IRAK-M expression.

    Article Snippet: Antibodies: GR (sc-8992), p65 (sc-8008, sc-372, sc-109), IRAK-M (sc-100389), α-Tubulin (sc-69969), β-actin (sc-8432), F4/80 (sc-26642), E-cadherin (sc-31020), Donkey anti-rabbit IgG-FITC (sc-2090) and bovine anti-goat IgG-TR (sc-2786) were purchased from Santa Cruz Biotechnology, IRAK-M (#2355) from ProSci, anti-rabbit HRP-linked antibody (#7074) and anti-mouse HRP-linked antibody (#7076) were from Cell Signaling. siRNAs; GR (cat# L-003424-00-0005, ON-TARGET plus Human NR3C1 (2908)-SMARTpool, p65 (cat# L-003533-00-0005, ON-TARGET plus Human RELA (5970) - SMARTpool) and control siRNA (cat# D-001810-10-05) were from Thermo Scientific Dharmacon, IRAK-M (IRAK3 (ID 11213) cat# SR307690 and control siRNA (cat# SR30004) were from OriGene.

    Techniques: Expressing

    ( a ) IRAK-M mRNA expression in human bronchial epithelial BEAS-2B cells treated with DEX (0.01, 0.1, 1, 10, 100 or 1,000 nM) for 1 h, followed by the stimulation of NTHi for 5 h. ( b ) IRAK-M mRNA expression in human lung epithelial A549 cells stimulated with DEX (100 nM) for 1 h, followed by NTHi for indicated time. ( c ) A549 cells were treated with DEX (100 nM) for 1 h, followed by NTHi for 12 h. IRAK-M protein was detected by anti-IRAK-M antibody. ( d ) IRAK-M protein expression was quantified from three independent experiments. ( e ) IRAK-M mRNA expression in primary NHBE cells treated with DEX (10, 100 or 1,000 nM) for 1 h and subjected to NTHi stimulation for 5 h. ( f ) Human peripheral blood CD14 + monocytes were differentiated to macrophages by culturing them with granulocyte–macrophage colony-stimulating factor for 7 days. IRAK-M mRNA was determined after the macrophages were treated with DEX (100 nM) for 1 h, followed by stimulation of NTHi for 5 h. ( g ) Mice were stimulated with DEX (1 mg kg −1 , intraperitoneally) for 2 h and intratracheally inoculated with NTHi (1 × 10 7 c.f.u.) for 24 h. The mRNA expression in alveolar macrophages was assessed by qPCR. ( h ) The IRAK-M mRNA expression in lung was assessed by qPCR. ( i ) Immunostaining of mouse lung with control IgG or anti-IRAK-M antibody by LSAB (Labelled Streptavidin Biotin) staining system (Scale bar, 50 μm; magnification, × 400). ( j ) Treatment-blind observers scored the IRAK-M expression from the histology results. ( k ) The lung sections were stained by using indicated antibodies. The arrows and arrowheads indicate the epithelial cells and macrophages merged with IRAK-M expression, respectively. (Scale bar, 50 μm; magnification, × 400. Data in a , b , d - h , n =3; j , n =6) are mean±s.d. * P< 0.05; t- test.

    Journal: Nature Communications

    Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

    doi: 10.1038/ncomms7062

    Figure Lengend Snippet: ( a ) IRAK-M mRNA expression in human bronchial epithelial BEAS-2B cells treated with DEX (0.01, 0.1, 1, 10, 100 or 1,000 nM) for 1 h, followed by the stimulation of NTHi for 5 h. ( b ) IRAK-M mRNA expression in human lung epithelial A549 cells stimulated with DEX (100 nM) for 1 h, followed by NTHi for indicated time. ( c ) A549 cells were treated with DEX (100 nM) for 1 h, followed by NTHi for 12 h. IRAK-M protein was detected by anti-IRAK-M antibody. ( d ) IRAK-M protein expression was quantified from three independent experiments. ( e ) IRAK-M mRNA expression in primary NHBE cells treated with DEX (10, 100 or 1,000 nM) for 1 h and subjected to NTHi stimulation for 5 h. ( f ) Human peripheral blood CD14 + monocytes were differentiated to macrophages by culturing them with granulocyte–macrophage colony-stimulating factor for 7 days. IRAK-M mRNA was determined after the macrophages were treated with DEX (100 nM) for 1 h, followed by stimulation of NTHi for 5 h. ( g ) Mice were stimulated with DEX (1 mg kg −1 , intraperitoneally) for 2 h and intratracheally inoculated with NTHi (1 × 10 7 c.f.u.) for 24 h. The mRNA expression in alveolar macrophages was assessed by qPCR. ( h ) The IRAK-M mRNA expression in lung was assessed by qPCR. ( i ) Immunostaining of mouse lung with control IgG or anti-IRAK-M antibody by LSAB (Labelled Streptavidin Biotin) staining system (Scale bar, 50 μm; magnification, × 400). ( j ) Treatment-blind observers scored the IRAK-M expression from the histology results. ( k ) The lung sections were stained by using indicated antibodies. The arrows and arrowheads indicate the epithelial cells and macrophages merged with IRAK-M expression, respectively. (Scale bar, 50 μm; magnification, × 400. Data in a , b , d - h , n =3; j , n =6) are mean±s.d. * P< 0.05; t- test.

    Article Snippet: The detection of IRAK-M protein was performed using rabbit anti-IRAK-M (ProSci, 2 μg ml −1 for Immunohistochemistry, 10 μg ml −1 for Immunofluorescence) and Donkey anti-rabbit-FITC (Santa Cruz Biotechnology, 5 μg ml −1 ) in the paraffin section of mouse lung tissue.

    Techniques: Expressing, Immunostaining, Staining

    ( a ) The proteins from BEAS-2B cells stably overexpressing IRAK-M and its control pcDNA3.1 (Mock) cells were detected by immunoblot with indicated antibodies. ( b ) The mRNA expression was detected by qPCR after the stable cells were treated with DEX (100 nM) and NTHi. ( c ) BEAS-2B cells were transfected with siRNA control or IRAK-M for 72 h. IRAK-M protein expression was detected by immunoblot with anti-IRAK-M antibodies (SC, Santa Cruz Biotechnology sc-100389; PS, ProSci #2355). ( d ) siRNA-transfected BEAS-2B cells were infected with NTHi followed by qPCR. ( e ) The mRNA level was calculated by following: (NTHi-induced mRNA at each concentration of DEX)/(NTHi-induced mRNA without DEX). Data in b , d , e , n =3) are mean±s.d. * P< 0.05; t- test.

    Journal: Nature Communications

    Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

    doi: 10.1038/ncomms7062

    Figure Lengend Snippet: ( a ) The proteins from BEAS-2B cells stably overexpressing IRAK-M and its control pcDNA3.1 (Mock) cells were detected by immunoblot with indicated antibodies. ( b ) The mRNA expression was detected by qPCR after the stable cells were treated with DEX (100 nM) and NTHi. ( c ) BEAS-2B cells were transfected with siRNA control or IRAK-M for 72 h. IRAK-M protein expression was detected by immunoblot with anti-IRAK-M antibodies (SC, Santa Cruz Biotechnology sc-100389; PS, ProSci #2355). ( d ) siRNA-transfected BEAS-2B cells were infected with NTHi followed by qPCR. ( e ) The mRNA level was calculated by following: (NTHi-induced mRNA at each concentration of DEX)/(NTHi-induced mRNA without DEX). Data in b , d , e , n =3) are mean±s.d. * P< 0.05; t- test.

    Article Snippet: The detection of IRAK-M protein was performed using rabbit anti-IRAK-M (ProSci, 2 μg ml −1 for Immunohistochemistry, 10 μg ml −1 for Immunofluorescence) and Donkey anti-rabbit-FITC (Santa Cruz Biotechnology, 5 μg ml −1 ) in the paraffin section of mouse lung tissue.

    Techniques: Stable Transfection, Western Blot, Expressing, Transfection, Infection, Concentration Assay

    WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 10 7 c.f.u. per lung) for 24 h. ( a ) The mRNA expression in the mouse lung tissue was analysed by qPCR. ( b ) The protein level of proinflammatory mediators in BAL fluid was determined by enzyme-linked immunosorbent assay. ( c ) The mRNA expression in alveolar macrophages was analysed by qPCR. Data in a , c , n =3; b , n =9 are mean±s.d. * P< 0.05, NS, non-significant; t- test.

    Journal: Nature Communications

    Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

    doi: 10.1038/ncomms7062

    Figure Lengend Snippet: WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 10 7 c.f.u. per lung) for 24 h. ( a ) The mRNA expression in the mouse lung tissue was analysed by qPCR. ( b ) The protein level of proinflammatory mediators in BAL fluid was determined by enzyme-linked immunosorbent assay. ( c ) The mRNA expression in alveolar macrophages was analysed by qPCR. Data in a , c , n =3; b , n =9 are mean±s.d. * P< 0.05, NS, non-significant; t- test.

    Article Snippet: The detection of IRAK-M protein was performed using rabbit anti-IRAK-M (ProSci, 2 μg ml −1 for Immunohistochemistry, 10 μg ml −1 for Immunofluorescence) and Donkey anti-rabbit-FITC (Santa Cruz Biotechnology, 5 μg ml −1 ) in the paraffin section of mouse lung tissue.

    Techniques: Expressing, Enzyme-linked Immunosorbent Assay

    ( a – e ) WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 10 7 c.f.u. per lung) for 24 h. ( a ) Haematoxylin and eosin (H&E) staining of lung tissues from mice (Scale bar, 200 μm, magnification, × 100 in large frame; Scale bar 50 μm, magnification, × 400 in inserted frame). ( b ) Blinded histopathologic scoring of lung inflammation was performed on H&E-stained lung sections in a grade 0–3. ( c ) The number of PMN cells from BAL fluid was counted using a haemocytometer under the microscope. ( d ) Bacterial loads (c.f.u.) in lung homogenates. ( e ) The mRNA expression of mBD4 in lung was measured by qPCR ( f ) WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with lethal dose of NTHi (5 × 10 8 c.f.u. per lung) for 7 days. Survival rate was monitored for indicated days. P values were determined by Kaplan–Meier survival analysis with GraphPad Prism 5.0. Data in b =8; c , e , n =3; d =10, f =20 are mean±s.d. * P< 0.05, NS, non-significant; t- test.

    Journal: Nature Communications

    Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

    doi: 10.1038/ncomms7062

    Figure Lengend Snippet: ( a – e ) WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 10 7 c.f.u. per lung) for 24 h. ( a ) Haematoxylin and eosin (H&E) staining of lung tissues from mice (Scale bar, 200 μm, magnification, × 100 in large frame; Scale bar 50 μm, magnification, × 400 in inserted frame). ( b ) Blinded histopathologic scoring of lung inflammation was performed on H&E-stained lung sections in a grade 0–3. ( c ) The number of PMN cells from BAL fluid was counted using a haemocytometer under the microscope. ( d ) Bacterial loads (c.f.u.) in lung homogenates. ( e ) The mRNA expression of mBD4 in lung was measured by qPCR ( f ) WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with lethal dose of NTHi (5 × 10 8 c.f.u. per lung) for 7 days. Survival rate was monitored for indicated days. P values were determined by Kaplan–Meier survival analysis with GraphPad Prism 5.0. Data in b =8; c , e , n =3; d =10, f =20 are mean±s.d. * P< 0.05, NS, non-significant; t- test.

    Article Snippet: The detection of IRAK-M protein was performed using rabbit anti-IRAK-M (ProSci, 2 μg ml −1 for Immunohistochemistry, 10 μg ml −1 for Immunofluorescence) and Donkey anti-rabbit-FITC (Santa Cruz Biotechnology, 5 μg ml −1 ) in the paraffin section of mouse lung tissue.

    Techniques: Staining, Microscopy, Expressing

    ( a ) Immunoblot shows that IRAK-M protein expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, TNF-α (10 ng ml −1 ), IL-1β (1 ng ml −1 ) or Pam3CSK4 (1 μg ml −1 ). ( b ) IRAK-M mRNA expression assessed by qPCR in A549 cells transfected with constitutive active form of IKKβ (IKKβ CA) with DEX (100 nM). ( c – f ), IRAK-M expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, IKKβ inhibitor (1 μM) ( c , d ) or RU486 (1 μM; e , f ). ( g ) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA GR. ( h ) BEAS-2B cells were treated with NTHi, DEX (100 nM) and actinomycin D, ActD (5 μg ml −1 ), followed by immunoblot. ( i ) The expression of IRAK-M mRNA in BEAS-2B cells treated with DEX (100 nM) and Compound A, CpdA (1 or 10 μM) followed by NTHi stimulation for 5 h. ( j ) Relative luciferase activity (RLA) of NF-κB is measured by luciferase assay. BEAS-2B cells transfected with NF-κB luciferase plasmid were treated with CpdA and NTHi. Data ( b , c , e , i , j , n =3) are mean±s.d. * P< 0.05, NS; t- test.

    Journal: Nature Communications

    Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

    doi: 10.1038/ncomms7062

    Figure Lengend Snippet: ( a ) Immunoblot shows that IRAK-M protein expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, TNF-α (10 ng ml −1 ), IL-1β (1 ng ml −1 ) or Pam3CSK4 (1 μg ml −1 ). ( b ) IRAK-M mRNA expression assessed by qPCR in A549 cells transfected with constitutive active form of IKKβ (IKKβ CA) with DEX (100 nM). ( c – f ), IRAK-M expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, IKKβ inhibitor (1 μM) ( c , d ) or RU486 (1 μM; e , f ). ( g ) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA GR. ( h ) BEAS-2B cells were treated with NTHi, DEX (100 nM) and actinomycin D, ActD (5 μg ml −1 ), followed by immunoblot. ( i ) The expression of IRAK-M mRNA in BEAS-2B cells treated with DEX (100 nM) and Compound A, CpdA (1 or 10 μM) followed by NTHi stimulation for 5 h. ( j ) Relative luciferase activity (RLA) of NF-κB is measured by luciferase assay. BEAS-2B cells transfected with NF-κB luciferase plasmid were treated with CpdA and NTHi. Data ( b , c , e , i , j , n =3) are mean±s.d. * P< 0.05, NS; t- test.

    Article Snippet: The detection of IRAK-M protein was performed using rabbit anti-IRAK-M (ProSci, 2 μg ml −1 for Immunohistochemistry, 10 μg ml −1 for Immunofluorescence) and Donkey anti-rabbit-FITC (Santa Cruz Biotechnology, 5 μg ml −1 ) in the paraffin section of mouse lung tissue.

    Techniques: Western Blot, Expressing, Transfection, Luciferase, Activity Assay, Plasmid Preparation

    ( a – c ) Relative luciferase activity (RLA) was measured after the transfection of IRAK-M promoter in pGL3 basic vector and treated with DEX (100 nM) and NTHi in BEAS-2B cells ( a , b ). ( c ) MEF cells were transfected with IRAK-M-luc (−500 bp), followed by DEX (1 μM) and NTHi stimulation. ( d ) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA p65. ( e , f ) ChIP assay in BEAS-2B cells treated with DEX and NTHi for 1 h. IRAK-M promoter regions from −38 to +39 bp for GRE, from −231 to −99 bp for κB site were amplified by qPCR. ( g ) Re-ChIP assay (detailed in Methods). PCR products were detected in agarose gel. ( h , i ) ChIP assay in BEAS-2B cells treated with IKKβ inhibitor (1 μM), RU486 (1 μM), DEX (100 nM) and NTHi. Data ( a – c , n =3; e , f , n =2) are mean±s.d. * P<0.05 ; t- test.

    Journal: Nature Communications

    Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

    doi: 10.1038/ncomms7062

    Figure Lengend Snippet: ( a – c ) Relative luciferase activity (RLA) was measured after the transfection of IRAK-M promoter in pGL3 basic vector and treated with DEX (100 nM) and NTHi in BEAS-2B cells ( a , b ). ( c ) MEF cells were transfected with IRAK-M-luc (−500 bp), followed by DEX (1 μM) and NTHi stimulation. ( d ) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA p65. ( e , f ) ChIP assay in BEAS-2B cells treated with DEX and NTHi for 1 h. IRAK-M promoter regions from −38 to +39 bp for GRE, from −231 to −99 bp for κB site were amplified by qPCR. ( g ) Re-ChIP assay (detailed in Methods). PCR products were detected in agarose gel. ( h , i ) ChIP assay in BEAS-2B cells treated with IKKβ inhibitor (1 μM), RU486 (1 μM), DEX (100 nM) and NTHi. Data ( a – c , n =3; e , f , n =2) are mean±s.d. * P<0.05 ; t- test.

    Article Snippet: The detection of IRAK-M protein was performed using rabbit anti-IRAK-M (ProSci, 2 μg ml −1 for Immunohistochemistry, 10 μg ml −1 for Immunofluorescence) and Donkey anti-rabbit-FITC (Santa Cruz Biotechnology, 5 μg ml −1 ) in the paraffin section of mouse lung tissue.

    Techniques: Luciferase, Activity Assay, Transfection, Plasmid Preparation, Western Blot, Expressing, Amplification, Agarose Gel Electrophoresis

    A schematic representation of suppression of inflammation by GCs via the upregulation of IRAK-M expression.

    Journal: Nature Communications

    Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

    doi: 10.1038/ncomms7062

    Figure Lengend Snippet: A schematic representation of suppression of inflammation by GCs via the upregulation of IRAK-M expression.

    Article Snippet: The detection of IRAK-M protein was performed using rabbit anti-IRAK-M (ProSci, 2 μg ml −1 for Immunohistochemistry, 10 μg ml −1 for Immunofluorescence) and Donkey anti-rabbit-FITC (Santa Cruz Biotechnology, 5 μg ml −1 ) in the paraffin section of mouse lung tissue.

    Techniques: Expressing

    ( a ) IRAK-M mRNA expression in human bronchial epithelial BEAS-2B cells treated with DEX (0.01, 0.1, 1, 10, 100 or 1,000 nM) for 1 h, followed by the stimulation of NTHi for 5 h. ( b ) IRAK-M mRNA expression in human lung epithelial A549 cells stimulated with DEX (100 nM) for 1 h, followed by NTHi for indicated time. ( c ) A549 cells were treated with DEX (100 nM) for 1 h, followed by NTHi for 12 h. IRAK-M protein was detected by anti-IRAK-M antibody. ( d ) IRAK-M protein expression was quantified from three independent experiments. ( e ) IRAK-M mRNA expression in primary NHBE cells treated with DEX (10, 100 or 1,000 nM) for 1 h and subjected to NTHi stimulation for 5 h. ( f ) Human peripheral blood CD14 + monocytes were differentiated to macrophages by culturing them with granulocyte–macrophage colony-stimulating factor for 7 days. IRAK-M mRNA was determined after the macrophages were treated with DEX (100 nM) for 1 h, followed by stimulation of NTHi for 5 h. ( g ) Mice were stimulated with DEX (1 mg kg −1 , intraperitoneally) for 2 h and intratracheally inoculated with NTHi (1 × 10 7 c.f.u.) for 24 h. The mRNA expression in alveolar macrophages was assessed by qPCR. ( h ) The IRAK-M mRNA expression in lung was assessed by qPCR. ( i ) Immunostaining of mouse lung with control IgG or anti-IRAK-M antibody by LSAB (Labelled Streptavidin Biotin) staining system (Scale bar, 50 μm; magnification, × 400). ( j ) Treatment-blind observers scored the IRAK-M expression from the histology results. ( k ) The lung sections were stained by using indicated antibodies. The arrows and arrowheads indicate the epithelial cells and macrophages merged with IRAK-M expression, respectively. (Scale bar, 50 μm; magnification, × 400. Data in a , b , d - h , n =3; j , n =6) are mean±s.d. * P< 0.05; t- test.

    Journal: Nature Communications

    Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

    doi: 10.1038/ncomms7062

    Figure Lengend Snippet: ( a ) IRAK-M mRNA expression in human bronchial epithelial BEAS-2B cells treated with DEX (0.01, 0.1, 1, 10, 100 or 1,000 nM) for 1 h, followed by the stimulation of NTHi for 5 h. ( b ) IRAK-M mRNA expression in human lung epithelial A549 cells stimulated with DEX (100 nM) for 1 h, followed by NTHi for indicated time. ( c ) A549 cells were treated with DEX (100 nM) for 1 h, followed by NTHi for 12 h. IRAK-M protein was detected by anti-IRAK-M antibody. ( d ) IRAK-M protein expression was quantified from three independent experiments. ( e ) IRAK-M mRNA expression in primary NHBE cells treated with DEX (10, 100 or 1,000 nM) for 1 h and subjected to NTHi stimulation for 5 h. ( f ) Human peripheral blood CD14 + monocytes were differentiated to macrophages by culturing them with granulocyte–macrophage colony-stimulating factor for 7 days. IRAK-M mRNA was determined after the macrophages were treated with DEX (100 nM) for 1 h, followed by stimulation of NTHi for 5 h. ( g ) Mice were stimulated with DEX (1 mg kg −1 , intraperitoneally) for 2 h and intratracheally inoculated with NTHi (1 × 10 7 c.f.u.) for 24 h. The mRNA expression in alveolar macrophages was assessed by qPCR. ( h ) The IRAK-M mRNA expression in lung was assessed by qPCR. ( i ) Immunostaining of mouse lung with control IgG or anti-IRAK-M antibody by LSAB (Labelled Streptavidin Biotin) staining system (Scale bar, 50 μm; magnification, × 400). ( j ) Treatment-blind observers scored the IRAK-M expression from the histology results. ( k ) The lung sections were stained by using indicated antibodies. The arrows and arrowheads indicate the epithelial cells and macrophages merged with IRAK-M expression, respectively. (Scale bar, 50 μm; magnification, × 400. Data in a , b , d - h , n =3; j , n =6) are mean±s.d. * P< 0.05; t- test.

    Article Snippet: Blocking IRAK-M peptide (ProSci, cat# 2355 P, 25 μg ml −1 ) was used for the negative control experiments.

    Techniques: Expressing, Immunostaining, Staining

    ( a ) The proteins from BEAS-2B cells stably overexpressing IRAK-M and its control pcDNA3.1 (Mock) cells were detected by immunoblot with indicated antibodies. ( b ) The mRNA expression was detected by qPCR after the stable cells were treated with DEX (100 nM) and NTHi. ( c ) BEAS-2B cells were transfected with siRNA control or IRAK-M for 72 h. IRAK-M protein expression was detected by immunoblot with anti-IRAK-M antibodies (SC, Santa Cruz Biotechnology sc-100389; PS, ProSci #2355). ( d ) siRNA-transfected BEAS-2B cells were infected with NTHi followed by qPCR. ( e ) The mRNA level was calculated by following: (NTHi-induced mRNA at each concentration of DEX)/(NTHi-induced mRNA without DEX). Data in b , d , e , n =3) are mean±s.d. * P< 0.05; t- test.

    Journal: Nature Communications

    Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

    doi: 10.1038/ncomms7062

    Figure Lengend Snippet: ( a ) The proteins from BEAS-2B cells stably overexpressing IRAK-M and its control pcDNA3.1 (Mock) cells were detected by immunoblot with indicated antibodies. ( b ) The mRNA expression was detected by qPCR after the stable cells were treated with DEX (100 nM) and NTHi. ( c ) BEAS-2B cells were transfected with siRNA control or IRAK-M for 72 h. IRAK-M protein expression was detected by immunoblot with anti-IRAK-M antibodies (SC, Santa Cruz Biotechnology sc-100389; PS, ProSci #2355). ( d ) siRNA-transfected BEAS-2B cells were infected with NTHi followed by qPCR. ( e ) The mRNA level was calculated by following: (NTHi-induced mRNA at each concentration of DEX)/(NTHi-induced mRNA without DEX). Data in b , d , e , n =3) are mean±s.d. * P< 0.05; t- test.

    Article Snippet: Blocking IRAK-M peptide (ProSci, cat# 2355 P, 25 μg ml −1 ) was used for the negative control experiments.

    Techniques: Stable Transfection, Western Blot, Expressing, Transfection, Infection, Concentration Assay

    WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 10 7 c.f.u. per lung) for 24 h. ( a ) The mRNA expression in the mouse lung tissue was analysed by qPCR. ( b ) The protein level of proinflammatory mediators in BAL fluid was determined by enzyme-linked immunosorbent assay. ( c ) The mRNA expression in alveolar macrophages was analysed by qPCR. Data in a , c , n =3; b , n =9 are mean±s.d. * P< 0.05, NS, non-significant; t- test.

    Journal: Nature Communications

    Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

    doi: 10.1038/ncomms7062

    Figure Lengend Snippet: WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 10 7 c.f.u. per lung) for 24 h. ( a ) The mRNA expression in the mouse lung tissue was analysed by qPCR. ( b ) The protein level of proinflammatory mediators in BAL fluid was determined by enzyme-linked immunosorbent assay. ( c ) The mRNA expression in alveolar macrophages was analysed by qPCR. Data in a , c , n =3; b , n =9 are mean±s.d. * P< 0.05, NS, non-significant; t- test.

    Article Snippet: Blocking IRAK-M peptide (ProSci, cat# 2355 P, 25 μg ml −1 ) was used for the negative control experiments.

    Techniques: Expressing, Enzyme-linked Immunosorbent Assay

    ( a – e ) WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 10 7 c.f.u. per lung) for 24 h. ( a ) Haematoxylin and eosin (H&E) staining of lung tissues from mice (Scale bar, 200 μm, magnification, × 100 in large frame; Scale bar 50 μm, magnification, × 400 in inserted frame). ( b ) Blinded histopathologic scoring of lung inflammation was performed on H&E-stained lung sections in a grade 0–3. ( c ) The number of PMN cells from BAL fluid was counted using a haemocytometer under the microscope. ( d ) Bacterial loads (c.f.u.) in lung homogenates. ( e ) The mRNA expression of mBD4 in lung was measured by qPCR ( f ) WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with lethal dose of NTHi (5 × 10 8 c.f.u. per lung) for 7 days. Survival rate was monitored for indicated days. P values were determined by Kaplan–Meier survival analysis with GraphPad Prism 5.0. Data in b =8; c , e , n =3; d =10, f =20 are mean±s.d. * P< 0.05, NS, non-significant; t- test.

    Journal: Nature Communications

    Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

    doi: 10.1038/ncomms7062

    Figure Lengend Snippet: ( a – e ) WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 10 7 c.f.u. per lung) for 24 h. ( a ) Haematoxylin and eosin (H&E) staining of lung tissues from mice (Scale bar, 200 μm, magnification, × 100 in large frame; Scale bar 50 μm, magnification, × 400 in inserted frame). ( b ) Blinded histopathologic scoring of lung inflammation was performed on H&E-stained lung sections in a grade 0–3. ( c ) The number of PMN cells from BAL fluid was counted using a haemocytometer under the microscope. ( d ) Bacterial loads (c.f.u.) in lung homogenates. ( e ) The mRNA expression of mBD4 in lung was measured by qPCR ( f ) WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with lethal dose of NTHi (5 × 10 8 c.f.u. per lung) for 7 days. Survival rate was monitored for indicated days. P values were determined by Kaplan–Meier survival analysis with GraphPad Prism 5.0. Data in b =8; c , e , n =3; d =10, f =20 are mean±s.d. * P< 0.05, NS, non-significant; t- test.

    Article Snippet: Blocking IRAK-M peptide (ProSci, cat# 2355 P, 25 μg ml −1 ) was used for the negative control experiments.

    Techniques: Staining, Microscopy, Expressing

    ( a ) Immunoblot shows that IRAK-M protein expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, TNF-α (10 ng ml −1 ), IL-1β (1 ng ml −1 ) or Pam3CSK4 (1 μg ml −1 ). ( b ) IRAK-M mRNA expression assessed by qPCR in A549 cells transfected with constitutive active form of IKKβ (IKKβ CA) with DEX (100 nM). ( c – f ), IRAK-M expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, IKKβ inhibitor (1 μM) ( c , d ) or RU486 (1 μM; e , f ). ( g ) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA GR. ( h ) BEAS-2B cells were treated with NTHi, DEX (100 nM) and actinomycin D, ActD (5 μg ml −1 ), followed by immunoblot. ( i ) The expression of IRAK-M mRNA in BEAS-2B cells treated with DEX (100 nM) and Compound A, CpdA (1 or 10 μM) followed by NTHi stimulation for 5 h. ( j ) Relative luciferase activity (RLA) of NF-κB is measured by luciferase assay. BEAS-2B cells transfected with NF-κB luciferase plasmid were treated with CpdA and NTHi. Data ( b , c , e , i , j , n =3) are mean±s.d. * P< 0.05, NS; t- test.

    Journal: Nature Communications

    Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

    doi: 10.1038/ncomms7062

    Figure Lengend Snippet: ( a ) Immunoblot shows that IRAK-M protein expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, TNF-α (10 ng ml −1 ), IL-1β (1 ng ml −1 ) or Pam3CSK4 (1 μg ml −1 ). ( b ) IRAK-M mRNA expression assessed by qPCR in A549 cells transfected with constitutive active form of IKKβ (IKKβ CA) with DEX (100 nM). ( c – f ), IRAK-M expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, IKKβ inhibitor (1 μM) ( c , d ) or RU486 (1 μM; e , f ). ( g ) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA GR. ( h ) BEAS-2B cells were treated with NTHi, DEX (100 nM) and actinomycin D, ActD (5 μg ml −1 ), followed by immunoblot. ( i ) The expression of IRAK-M mRNA in BEAS-2B cells treated with DEX (100 nM) and Compound A, CpdA (1 or 10 μM) followed by NTHi stimulation for 5 h. ( j ) Relative luciferase activity (RLA) of NF-κB is measured by luciferase assay. BEAS-2B cells transfected with NF-κB luciferase plasmid were treated with CpdA and NTHi. Data ( b , c , e , i , j , n =3) are mean±s.d. * P< 0.05, NS; t- test.

    Article Snippet: Blocking IRAK-M peptide (ProSci, cat# 2355 P, 25 μg ml −1 ) was used for the negative control experiments.

    Techniques: Western Blot, Expressing, Transfection, Luciferase, Activity Assay, Plasmid Preparation

    ( a – c ) Relative luciferase activity (RLA) was measured after the transfection of IRAK-M promoter in pGL3 basic vector and treated with DEX (100 nM) and NTHi in BEAS-2B cells ( a , b ). ( c ) MEF cells were transfected with IRAK-M-luc (−500 bp), followed by DEX (1 μM) and NTHi stimulation. ( d ) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA p65. ( e , f ) ChIP assay in BEAS-2B cells treated with DEX and NTHi for 1 h. IRAK-M promoter regions from −38 to +39 bp for GRE, from −231 to −99 bp for κB site were amplified by qPCR. ( g ) Re-ChIP assay (detailed in Methods). PCR products were detected in agarose gel. ( h , i ) ChIP assay in BEAS-2B cells treated with IKKβ inhibitor (1 μM), RU486 (1 μM), DEX (100 nM) and NTHi. Data ( a – c , n =3; e , f , n =2) are mean±s.d. * P<0.05 ; t- test.

    Journal: Nature Communications

    Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

    doi: 10.1038/ncomms7062

    Figure Lengend Snippet: ( a – c ) Relative luciferase activity (RLA) was measured after the transfection of IRAK-M promoter in pGL3 basic vector and treated with DEX (100 nM) and NTHi in BEAS-2B cells ( a , b ). ( c ) MEF cells were transfected with IRAK-M-luc (−500 bp), followed by DEX (1 μM) and NTHi stimulation. ( d ) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA p65. ( e , f ) ChIP assay in BEAS-2B cells treated with DEX and NTHi for 1 h. IRAK-M promoter regions from −38 to +39 bp for GRE, from −231 to −99 bp for κB site were amplified by qPCR. ( g ) Re-ChIP assay (detailed in Methods). PCR products were detected in agarose gel. ( h , i ) ChIP assay in BEAS-2B cells treated with IKKβ inhibitor (1 μM), RU486 (1 μM), DEX (100 nM) and NTHi. Data ( a – c , n =3; e , f , n =2) are mean±s.d. * P<0.05 ; t- test.

    Article Snippet: Blocking IRAK-M peptide (ProSci, cat# 2355 P, 25 μg ml −1 ) was used for the negative control experiments.

    Techniques: Luciferase, Activity Assay, Transfection, Plasmid Preparation, Western Blot, Expressing, Amplification, Agarose Gel Electrophoresis

    A schematic representation of suppression of inflammation by GCs via the upregulation of IRAK-M expression.

    Journal: Nature Communications

    Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

    doi: 10.1038/ncomms7062

    Figure Lengend Snippet: A schematic representation of suppression of inflammation by GCs via the upregulation of IRAK-M expression.

    Article Snippet: Blocking IRAK-M peptide (ProSci, cat# 2355 P, 25 μg ml −1 ) was used for the negative control experiments.

    Techniques: Expressing

    ( a ) IRAK-M mRNA expression in human bronchial epithelial BEAS-2B cells treated with DEX (0.01, 0.1, 1, 10, 100 or 1,000 nM) for 1 h, followed by the stimulation of NTHi for 5 h. ( b ) IRAK-M mRNA expression in human lung epithelial A549 cells stimulated with DEX (100 nM) for 1 h, followed by NTHi for indicated time. ( c ) A549 cells were treated with DEX (100 nM) for 1 h, followed by NTHi for 12 h. IRAK-M protein was detected by anti-IRAK-M antibody. ( d ) IRAK-M protein expression was quantified from three independent experiments. ( e ) IRAK-M mRNA expression in primary NHBE cells treated with DEX (10, 100 or 1,000 nM) for 1 h and subjected to NTHi stimulation for 5 h. ( f ) Human peripheral blood CD14 + monocytes were differentiated to macrophages by culturing them with granulocyte–macrophage colony-stimulating factor for 7 days. IRAK-M mRNA was determined after the macrophages were treated with DEX (100 nM) for 1 h, followed by stimulation of NTHi for 5 h. ( g ) Mice were stimulated with DEX (1 mg kg −1 , intraperitoneally) for 2 h and intratracheally inoculated with NTHi (1 × 10 7 c.f.u.) for 24 h. The mRNA expression in alveolar macrophages was assessed by qPCR. ( h ) The IRAK-M mRNA expression in lung was assessed by qPCR. ( i ) Immunostaining of mouse lung with control IgG or anti-IRAK-M antibody by LSAB (Labelled Streptavidin Biotin) staining system (Scale bar, 50 μm; magnification, × 400). ( j ) Treatment-blind observers scored the IRAK-M expression from the histology results. ( k ) The lung sections were stained by using indicated antibodies. The arrows and arrowheads indicate the epithelial cells and macrophages merged with IRAK-M expression, respectively. (Scale bar, 50 μm; magnification, × 400. Data in a , b , d - h , n =3; j , n =6) are mean±s.d. * P< 0.05; t- test.

    Journal: Nature Communications

    Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

    doi: 10.1038/ncomms7062

    Figure Lengend Snippet: ( a ) IRAK-M mRNA expression in human bronchial epithelial BEAS-2B cells treated with DEX (0.01, 0.1, 1, 10, 100 or 1,000 nM) for 1 h, followed by the stimulation of NTHi for 5 h. ( b ) IRAK-M mRNA expression in human lung epithelial A549 cells stimulated with DEX (100 nM) for 1 h, followed by NTHi for indicated time. ( c ) A549 cells were treated with DEX (100 nM) for 1 h, followed by NTHi for 12 h. IRAK-M protein was detected by anti-IRAK-M antibody. ( d ) IRAK-M protein expression was quantified from three independent experiments. ( e ) IRAK-M mRNA expression in primary NHBE cells treated with DEX (10, 100 or 1,000 nM) for 1 h and subjected to NTHi stimulation for 5 h. ( f ) Human peripheral blood CD14 + monocytes were differentiated to macrophages by culturing them with granulocyte–macrophage colony-stimulating factor for 7 days. IRAK-M mRNA was determined after the macrophages were treated with DEX (100 nM) for 1 h, followed by stimulation of NTHi for 5 h. ( g ) Mice were stimulated with DEX (1 mg kg −1 , intraperitoneally) for 2 h and intratracheally inoculated with NTHi (1 × 10 7 c.f.u.) for 24 h. The mRNA expression in alveolar macrophages was assessed by qPCR. ( h ) The IRAK-M mRNA expression in lung was assessed by qPCR. ( i ) Immunostaining of mouse lung with control IgG or anti-IRAK-M antibody by LSAB (Labelled Streptavidin Biotin) staining system (Scale bar, 50 μm; magnification, × 400). ( j ) Treatment-blind observers scored the IRAK-M expression from the histology results. ( k ) The lung sections were stained by using indicated antibodies. The arrows and arrowheads indicate the epithelial cells and macrophages merged with IRAK-M expression, respectively. (Scale bar, 50 μm; magnification, × 400. Data in a , b , d - h , n =3; j , n =6) are mean±s.d. * P< 0.05; t- test.

    Article Snippet: The detection of IRAK-M protein was performed using rabbit anti-IRAK-M (ProSci, 2 μg ml −1 for Immunohistochemistry, 10 μg ml −1 for Immunofluorescence) and Donkey anti-rabbit-FITC (Santa Cruz Biotechnology, 5 μg ml −1 ) in the paraffin section of mouse lung tissue.

    Techniques: Expressing, Immunostaining, Staining

    ( a ) The proteins from BEAS-2B cells stably overexpressing IRAK-M and its control pcDNA3.1 (Mock) cells were detected by immunoblot with indicated antibodies. ( b ) The mRNA expression was detected by qPCR after the stable cells were treated with DEX (100 nM) and NTHi. ( c ) BEAS-2B cells were transfected with siRNA control or IRAK-M for 72 h. IRAK-M protein expression was detected by immunoblot with anti-IRAK-M antibodies (SC, Santa Cruz Biotechnology sc-100389; PS, ProSci #2355). ( d ) siRNA-transfected BEAS-2B cells were infected with NTHi followed by qPCR. ( e ) The mRNA level was calculated by following: (NTHi-induced mRNA at each concentration of DEX)/(NTHi-induced mRNA without DEX). Data in b , d , e , n =3) are mean±s.d. * P< 0.05; t- test.

    Journal: Nature Communications

    Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

    doi: 10.1038/ncomms7062

    Figure Lengend Snippet: ( a ) The proteins from BEAS-2B cells stably overexpressing IRAK-M and its control pcDNA3.1 (Mock) cells were detected by immunoblot with indicated antibodies. ( b ) The mRNA expression was detected by qPCR after the stable cells were treated with DEX (100 nM) and NTHi. ( c ) BEAS-2B cells were transfected with siRNA control or IRAK-M for 72 h. IRAK-M protein expression was detected by immunoblot with anti-IRAK-M antibodies (SC, Santa Cruz Biotechnology sc-100389; PS, ProSci #2355). ( d ) siRNA-transfected BEAS-2B cells were infected with NTHi followed by qPCR. ( e ) The mRNA level was calculated by following: (NTHi-induced mRNA at each concentration of DEX)/(NTHi-induced mRNA without DEX). Data in b , d , e , n =3) are mean±s.d. * P< 0.05; t- test.

    Article Snippet: The detection of IRAK-M protein was performed using rabbit anti-IRAK-M (ProSci, 2 μg ml −1 for Immunohistochemistry, 10 μg ml −1 for Immunofluorescence) and Donkey anti-rabbit-FITC (Santa Cruz Biotechnology, 5 μg ml −1 ) in the paraffin section of mouse lung tissue.

    Techniques: Stable Transfection, Western Blot, Expressing, Transfection, Infection, Concentration Assay

    WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 10 7 c.f.u. per lung) for 24 h. ( a ) The mRNA expression in the mouse lung tissue was analysed by qPCR. ( b ) The protein level of proinflammatory mediators in BAL fluid was determined by enzyme-linked immunosorbent assay. ( c ) The mRNA expression in alveolar macrophages was analysed by qPCR. Data in a , c , n =3; b , n =9 are mean±s.d. * P< 0.05, NS, non-significant; t- test.

    Journal: Nature Communications

    Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

    doi: 10.1038/ncomms7062

    Figure Lengend Snippet: WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 10 7 c.f.u. per lung) for 24 h. ( a ) The mRNA expression in the mouse lung tissue was analysed by qPCR. ( b ) The protein level of proinflammatory mediators in BAL fluid was determined by enzyme-linked immunosorbent assay. ( c ) The mRNA expression in alveolar macrophages was analysed by qPCR. Data in a , c , n =3; b , n =9 are mean±s.d. * P< 0.05, NS, non-significant; t- test.

    Article Snippet: The detection of IRAK-M protein was performed using rabbit anti-IRAK-M (ProSci, 2 μg ml −1 for Immunohistochemistry, 10 μg ml −1 for Immunofluorescence) and Donkey anti-rabbit-FITC (Santa Cruz Biotechnology, 5 μg ml −1 ) in the paraffin section of mouse lung tissue.

    Techniques: Expressing, Enzyme-linked Immunosorbent Assay

    ( a – e ) WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 10 7 c.f.u. per lung) for 24 h. ( a ) Haematoxylin and eosin (H&E) staining of lung tissues from mice (Scale bar, 200 μm, magnification, × 100 in large frame; Scale bar 50 μm, magnification, × 400 in inserted frame). ( b ) Blinded histopathologic scoring of lung inflammation was performed on H&E-stained lung sections in a grade 0–3. ( c ) The number of PMN cells from BAL fluid was counted using a haemocytometer under the microscope. ( d ) Bacterial loads (c.f.u.) in lung homogenates. ( e ) The mRNA expression of mBD4 in lung was measured by qPCR ( f ) WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with lethal dose of NTHi (5 × 10 8 c.f.u. per lung) for 7 days. Survival rate was monitored for indicated days. P values were determined by Kaplan–Meier survival analysis with GraphPad Prism 5.0. Data in b =8; c , e , n =3; d =10, f =20 are mean±s.d. * P< 0.05, NS, non-significant; t- test.

    Journal: Nature Communications

    Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

    doi: 10.1038/ncomms7062

    Figure Lengend Snippet: ( a – e ) WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 × 10 7 c.f.u. per lung) for 24 h. ( a ) Haematoxylin and eosin (H&E) staining of lung tissues from mice (Scale bar, 200 μm, magnification, × 100 in large frame; Scale bar 50 μm, magnification, × 400 in inserted frame). ( b ) Blinded histopathologic scoring of lung inflammation was performed on H&E-stained lung sections in a grade 0–3. ( c ) The number of PMN cells from BAL fluid was counted using a haemocytometer under the microscope. ( d ) Bacterial loads (c.f.u.) in lung homogenates. ( e ) The mRNA expression of mBD4 in lung was measured by qPCR ( f ) WT and IRAK-M-deficient mice were inoculated with DEX (1 mg kg −1 , intraperitoneally) for 2 h, followed by intratracheal inoculation with lethal dose of NTHi (5 × 10 8 c.f.u. per lung) for 7 days. Survival rate was monitored for indicated days. P values were determined by Kaplan–Meier survival analysis with GraphPad Prism 5.0. Data in b =8; c , e , n =3; d =10, f =20 are mean±s.d. * P< 0.05, NS, non-significant; t- test.

    Article Snippet: The detection of IRAK-M protein was performed using rabbit anti-IRAK-M (ProSci, 2 μg ml −1 for Immunohistochemistry, 10 μg ml −1 for Immunofluorescence) and Donkey anti-rabbit-FITC (Santa Cruz Biotechnology, 5 μg ml −1 ) in the paraffin section of mouse lung tissue.

    Techniques: Staining, Microscopy, Expressing

    ( a ) Immunoblot shows that IRAK-M protein expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, TNF-α (10 ng ml −1 ), IL-1β (1 ng ml −1 ) or Pam3CSK4 (1 μg ml −1 ). ( b ) IRAK-M mRNA expression assessed by qPCR in A549 cells transfected with constitutive active form of IKKβ (IKKβ CA) with DEX (100 nM). ( c – f ), IRAK-M expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, IKKβ inhibitor (1 μM) ( c , d ) or RU486 (1 μM; e , f ). ( g ) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA GR. ( h ) BEAS-2B cells were treated with NTHi, DEX (100 nM) and actinomycin D, ActD (5 μg ml −1 ), followed by immunoblot. ( i ) The expression of IRAK-M mRNA in BEAS-2B cells treated with DEX (100 nM) and Compound A, CpdA (1 or 10 μM) followed by NTHi stimulation for 5 h. ( j ) Relative luciferase activity (RLA) of NF-κB is measured by luciferase assay. BEAS-2B cells transfected with NF-κB luciferase plasmid were treated with CpdA and NTHi. Data ( b , c , e , i , j , n =3) are mean±s.d. * P< 0.05, NS; t- test.

    Journal: Nature Communications

    Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

    doi: 10.1038/ncomms7062

    Figure Lengend Snippet: ( a ) Immunoblot shows that IRAK-M protein expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, TNF-α (10 ng ml −1 ), IL-1β (1 ng ml −1 ) or Pam3CSK4 (1 μg ml −1 ). ( b ) IRAK-M mRNA expression assessed by qPCR in A549 cells transfected with constitutive active form of IKKβ (IKKβ CA) with DEX (100 nM). ( c – f ), IRAK-M expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, IKKβ inhibitor (1 μM) ( c , d ) or RU486 (1 μM; e , f ). ( g ) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA GR. ( h ) BEAS-2B cells were treated with NTHi, DEX (100 nM) and actinomycin D, ActD (5 μg ml −1 ), followed by immunoblot. ( i ) The expression of IRAK-M mRNA in BEAS-2B cells treated with DEX (100 nM) and Compound A, CpdA (1 or 10 μM) followed by NTHi stimulation for 5 h. ( j ) Relative luciferase activity (RLA) of NF-κB is measured by luciferase assay. BEAS-2B cells transfected with NF-κB luciferase plasmid were treated with CpdA and NTHi. Data ( b , c , e , i , j , n =3) are mean±s.d. * P< 0.05, NS; t- test.

    Article Snippet: The detection of IRAK-M protein was performed using rabbit anti-IRAK-M (ProSci, 2 μg ml −1 for Immunohistochemistry, 10 μg ml −1 for Immunofluorescence) and Donkey anti-rabbit-FITC (Santa Cruz Biotechnology, 5 μg ml −1 ) in the paraffin section of mouse lung tissue.

    Techniques: Western Blot, Expressing, Transfection, Luciferase, Activity Assay, Plasmid Preparation

    ( a – c ) Relative luciferase activity (RLA) was measured after the transfection of IRAK-M promoter in pGL3 basic vector and treated with DEX (100 nM) and NTHi in BEAS-2B cells ( a , b ). ( c ) MEF cells were transfected with IRAK-M-luc (−500 bp), followed by DEX (1 μM) and NTHi stimulation. ( d ) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA p65. ( e , f ) ChIP assay in BEAS-2B cells treated with DEX and NTHi for 1 h. IRAK-M promoter regions from −38 to +39 bp for GRE, from −231 to −99 bp for κB site were amplified by qPCR. ( g ) Re-ChIP assay (detailed in Methods). PCR products were detected in agarose gel. ( h , i ) ChIP assay in BEAS-2B cells treated with IKKβ inhibitor (1 μM), RU486 (1 μM), DEX (100 nM) and NTHi. Data ( a – c , n =3; e , f , n =2) are mean±s.d. * P<0.05 ; t- test.

    Journal: Nature Communications

    Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

    doi: 10.1038/ncomms7062

    Figure Lengend Snippet: ( a – c ) Relative luciferase activity (RLA) was measured after the transfection of IRAK-M promoter in pGL3 basic vector and treated with DEX (100 nM) and NTHi in BEAS-2B cells ( a , b ). ( c ) MEF cells were transfected with IRAK-M-luc (−500 bp), followed by DEX (1 μM) and NTHi stimulation. ( d ) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA p65. ( e , f ) ChIP assay in BEAS-2B cells treated with DEX and NTHi for 1 h. IRAK-M promoter regions from −38 to +39 bp for GRE, from −231 to −99 bp for κB site were amplified by qPCR. ( g ) Re-ChIP assay (detailed in Methods). PCR products were detected in agarose gel. ( h , i ) ChIP assay in BEAS-2B cells treated with IKKβ inhibitor (1 μM), RU486 (1 μM), DEX (100 nM) and NTHi. Data ( a – c , n =3; e , f , n =2) are mean±s.d. * P<0.05 ; t- test.

    Article Snippet: The detection of IRAK-M protein was performed using rabbit anti-IRAK-M (ProSci, 2 μg ml −1 for Immunohistochemistry, 10 μg ml −1 for Immunofluorescence) and Donkey anti-rabbit-FITC (Santa Cruz Biotechnology, 5 μg ml −1 ) in the paraffin section of mouse lung tissue.

    Techniques: Luciferase, Activity Assay, Transfection, Plasmid Preparation, Western Blot, Expressing, Amplification, Agarose Gel Electrophoresis

    A schematic representation of suppression of inflammation by GCs via the upregulation of IRAK-M expression.

    Journal: Nature Communications

    Article Title: Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

    doi: 10.1038/ncomms7062

    Figure Lengend Snippet: A schematic representation of suppression of inflammation by GCs via the upregulation of IRAK-M expression.

    Article Snippet: The detection of IRAK-M protein was performed using rabbit anti-IRAK-M (ProSci, 2 μg ml −1 for Immunohistochemistry, 10 μg ml −1 for Immunofluorescence) and Donkey anti-rabbit-FITC (Santa Cruz Biotechnology, 5 μg ml −1 ) in the paraffin section of mouse lung tissue.

    Techniques: Expressing