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Millipore expand high fidelity pcr system
Mechanisms of <t>GILZ</t> downregulation upon TLR activation. (A,B) AMs pretreated with BAY-11-7082 (BAY82, 5 μM), BAY-11-7085 (BAY85, 5 μM), or the solvent control DMSO (0.1%) for 1 h, followed by treatment with LPS (100 ng/mL), Pam 3 CSK 4 (Pam, 1 μg/mL), Poly(I:C) (PIC, 10 μg/mL), or medium (Co) for 4 h. GILZ expression was determined by Western blot. Tubulin served as a loading control. (A) Representative blot. (B) GILZ signal intensities were quantified and normalized to tubulin values ( n = 7). Values for unstimulated DMSO controls were set as 100%. (C,D) After preincubation with BAY-11-7082 or BAY-11-7085 (5 μM, 1 h), solvent (0.1% DMSO) or medium only (Co), AMs were treated with LPS (100 ng/mL) for 2 h. GILZ and ZFP36 mRNA expression was determined by <t>qRT-PCR</t> using ACTB as a housekeeping gene ( n = 3, duplicates). (E) AMs were either left untreated (Co) or treated with LPS (100 ng/mL), Pam 3 CSK 4 (Pam, 1 μg/mL), or Poly(I:C) (PIC, 10 μg/mL) for 4 h. TTP levels were determined by Western blot using tubulin as a loading control. GILZ signal intensities were normalized to tubulin and are shown as a percentage of untreated cells ( n = 2, triplicates). (F) The number of miRNAs predicted to target GILZ, CXCL8, IL6 , and TNF was assessed via the microRNA Data Integration Portal (mirDIP, accession date 02/02/2018). (G) AMs were either left untreated (Co) or treated with Poly(I:C) (PIC, 10 μg/mL), aurintricarboxylic acid (ATA, 25 μM), or a combination of both for 8 h. GILZ expression was quantified by Western blot. Signal intensities were normalized to tubulin and expressed as a percentage of untreated cells ( n = 3). * p
Expand High Fidelity Pcr System, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Amplified Host Defense by Toll-Like Receptor-Mediated Downregulation of the Glucocorticoid-Induced Leucine Zipper (GILZ) in Macrophages"

Article Title: Amplified Host Defense by Toll-Like Receptor-Mediated Downregulation of the Glucocorticoid-Induced Leucine Zipper (GILZ) in Macrophages

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.03111

Mechanisms of GILZ downregulation upon TLR activation. (A,B) AMs pretreated with BAY-11-7082 (BAY82, 5 μM), BAY-11-7085 (BAY85, 5 μM), or the solvent control DMSO (0.1%) for 1 h, followed by treatment with LPS (100 ng/mL), Pam 3 CSK 4 (Pam, 1 μg/mL), Poly(I:C) (PIC, 10 μg/mL), or medium (Co) for 4 h. GILZ expression was determined by Western blot. Tubulin served as a loading control. (A) Representative blot. (B) GILZ signal intensities were quantified and normalized to tubulin values ( n = 7). Values for unstimulated DMSO controls were set as 100%. (C,D) After preincubation with BAY-11-7082 or BAY-11-7085 (5 μM, 1 h), solvent (0.1% DMSO) or medium only (Co), AMs were treated with LPS (100 ng/mL) for 2 h. GILZ and ZFP36 mRNA expression was determined by qRT-PCR using ACTB as a housekeeping gene ( n = 3, duplicates). (E) AMs were either left untreated (Co) or treated with LPS (100 ng/mL), Pam 3 CSK 4 (Pam, 1 μg/mL), or Poly(I:C) (PIC, 10 μg/mL) for 4 h. TTP levels were determined by Western blot using tubulin as a loading control. GILZ signal intensities were normalized to tubulin and are shown as a percentage of untreated cells ( n = 2, triplicates). (F) The number of miRNAs predicted to target GILZ, CXCL8, IL6 , and TNF was assessed via the microRNA Data Integration Portal (mirDIP, accession date 02/02/2018). (G) AMs were either left untreated (Co) or treated with Poly(I:C) (PIC, 10 μg/mL), aurintricarboxylic acid (ATA, 25 μM), or a combination of both for 8 h. GILZ expression was quantified by Western blot. Signal intensities were normalized to tubulin and expressed as a percentage of untreated cells ( n = 3). * p
Figure Legend Snippet: Mechanisms of GILZ downregulation upon TLR activation. (A,B) AMs pretreated with BAY-11-7082 (BAY82, 5 μM), BAY-11-7085 (BAY85, 5 μM), or the solvent control DMSO (0.1%) for 1 h, followed by treatment with LPS (100 ng/mL), Pam 3 CSK 4 (Pam, 1 μg/mL), Poly(I:C) (PIC, 10 μg/mL), or medium (Co) for 4 h. GILZ expression was determined by Western blot. Tubulin served as a loading control. (A) Representative blot. (B) GILZ signal intensities were quantified and normalized to tubulin values ( n = 7). Values for unstimulated DMSO controls were set as 100%. (C,D) After preincubation with BAY-11-7082 or BAY-11-7085 (5 μM, 1 h), solvent (0.1% DMSO) or medium only (Co), AMs were treated with LPS (100 ng/mL) for 2 h. GILZ and ZFP36 mRNA expression was determined by qRT-PCR using ACTB as a housekeeping gene ( n = 3, duplicates). (E) AMs were either left untreated (Co) or treated with LPS (100 ng/mL), Pam 3 CSK 4 (Pam, 1 μg/mL), or Poly(I:C) (PIC, 10 μg/mL) for 4 h. TTP levels were determined by Western blot using tubulin as a loading control. GILZ signal intensities were normalized to tubulin and are shown as a percentage of untreated cells ( n = 2, triplicates). (F) The number of miRNAs predicted to target GILZ, CXCL8, IL6 , and TNF was assessed via the microRNA Data Integration Portal (mirDIP, accession date 02/02/2018). (G) AMs were either left untreated (Co) or treated with Poly(I:C) (PIC, 10 μg/mL), aurintricarboxylic acid (ATA, 25 μM), or a combination of both for 8 h. GILZ expression was quantified by Western blot. Signal intensities were normalized to tubulin and expressed as a percentage of untreated cells ( n = 3). * p

Techniques Used: Activation Assay, Affinity Magnetic Separation, Expressing, Western Blot, Quantitative RT-PCR

2) Product Images from "Amplified Host Defense by Toll-Like Receptor-Mediated Downregulation of the Glucocorticoid-Induced Leucine Zipper (GILZ) in Macrophages"

Article Title: Amplified Host Defense by Toll-Like Receptor-Mediated Downregulation of the Glucocorticoid-Induced Leucine Zipper (GILZ) in Macrophages

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.03111

Mechanisms of GILZ downregulation upon TLR activation. (A,B) AMs pretreated with BAY-11-7082 (BAY82, 5 μM), BAY-11-7085 (BAY85, 5 μM), or the solvent control DMSO (0.1%) for 1 h, followed by treatment with LPS (100 ng/mL), Pam 3 CSK 4 (Pam, 1 μg/mL), Poly(I:C) (PIC, 10 μg/mL), or medium (Co) for 4 h. GILZ expression was determined by Western blot. Tubulin served as a loading control. (A) Representative blot. (B) GILZ signal intensities were quantified and normalized to tubulin values ( n = 7). Values for unstimulated DMSO controls were set as 100%. (C,D) After preincubation with BAY-11-7082 or BAY-11-7085 (5 μM, 1 h), solvent (0.1% DMSO) or medium only (Co), AMs were treated with LPS (100 ng/mL) for 2 h. GILZ and ZFP36 mRNA expression was determined by qRT-PCR using ACTB as a housekeeping gene ( n = 3, duplicates). (E) AMs were either left untreated (Co) or treated with LPS (100 ng/mL), Pam 3 CSK 4 (Pam, 1 μg/mL), or Poly(I:C) (PIC, 10 μg/mL) for 4 h. TTP levels were determined by Western blot using tubulin as a loading control. GILZ signal intensities were normalized to tubulin and are shown as a percentage of untreated cells ( n = 2, triplicates). (F) The number of miRNAs predicted to target GILZ, CXCL8, IL6 , and TNF was assessed via the microRNA Data Integration Portal (mirDIP, accession date 02/02/2018). (G) AMs were either left untreated (Co) or treated with Poly(I:C) (PIC, 10 μg/mL), aurintricarboxylic acid (ATA, 25 μM), or a combination of both for 8 h. GILZ expression was quantified by Western blot. Signal intensities were normalized to tubulin and expressed as a percentage of untreated cells ( n = 3). * p
Figure Legend Snippet: Mechanisms of GILZ downregulation upon TLR activation. (A,B) AMs pretreated with BAY-11-7082 (BAY82, 5 μM), BAY-11-7085 (BAY85, 5 μM), or the solvent control DMSO (0.1%) for 1 h, followed by treatment with LPS (100 ng/mL), Pam 3 CSK 4 (Pam, 1 μg/mL), Poly(I:C) (PIC, 10 μg/mL), or medium (Co) for 4 h. GILZ expression was determined by Western blot. Tubulin served as a loading control. (A) Representative blot. (B) GILZ signal intensities were quantified and normalized to tubulin values ( n = 7). Values for unstimulated DMSO controls were set as 100%. (C,D) After preincubation with BAY-11-7082 or BAY-11-7085 (5 μM, 1 h), solvent (0.1% DMSO) or medium only (Co), AMs were treated with LPS (100 ng/mL) for 2 h. GILZ and ZFP36 mRNA expression was determined by qRT-PCR using ACTB as a housekeeping gene ( n = 3, duplicates). (E) AMs were either left untreated (Co) or treated with LPS (100 ng/mL), Pam 3 CSK 4 (Pam, 1 μg/mL), or Poly(I:C) (PIC, 10 μg/mL) for 4 h. TTP levels were determined by Western blot using tubulin as a loading control. GILZ signal intensities were normalized to tubulin and are shown as a percentage of untreated cells ( n = 2, triplicates). (F) The number of miRNAs predicted to target GILZ, CXCL8, IL6 , and TNF was assessed via the microRNA Data Integration Portal (mirDIP, accession date 02/02/2018). (G) AMs were either left untreated (Co) or treated with Poly(I:C) (PIC, 10 μg/mL), aurintricarboxylic acid (ATA, 25 μM), or a combination of both for 8 h. GILZ expression was quantified by Western blot. Signal intensities were normalized to tubulin and expressed as a percentage of untreated cells ( n = 3). * p

Techniques Used: Activation Assay, Affinity Magnetic Separation, Expressing, Western Blot, Quantitative RT-PCR

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Amplification:

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Polymerase Chain Reaction:

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Nested PCR:

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Activated Clotting Time Assay:

Article Title: Amplified Host Defense by Toll-Like Receptor-Mediated Downregulation of the Glucocorticoid-Induced Leucine Zipper (GILZ) in Macrophages
Article Snippet: GILZ 3′UTR-luciferase reporter constructs were generated by coupling the GILZ 3′UTR sequence to a firefly luciferase reporter gene ( ). .. Human GILZ 3′UTR cDNA was amplified using the Expand High fidelity PCR System (Sigma-Aldrich, # 11732641001) and the following primers: 5′-GCC TAC TAG TGC AGA GCC ACT AAA CTT G-3′ and 5′-AAT AGA GCT CAC TCT CAC AAA ACC CGC TAC-3′. .. The SacI/SpeI digested PCR product was inserted into the respective cloning site of pMIR-REPORT (Ambion, #AM5795).

Ligation:

Article Title: Human Nup98 regulates the localization and activity of DExH/D-box helicase DHX9
Article Snippet: The ligation reaction was digested with DpnII (New England Biolabs R0543S) in a 50 µl reaction for 1 hr. .. Ten microliters of the DpnII digested ligation sample was amplified by PCR with Expand High Fidelity PCR System as described by the manufacturer (Sigma-Aldrich 11732641001). .. The resulting amplified DNA was purified with QIAquick PCR Purification Kit (QIAgen 28104) and used as template for real-time PCRs, as described above.

Sequencing:

Article Title: In Vitro and In Vivo Characterization of a Pigeon Paramyxovirus Type 1 Isolated from Domestic Pigeons in Victoria, Australia 2011
Article Snippet: .. Whole genome sequencing was performed using the Illumina MiSeq platform (Illumina, San Diego, CA, USA). cDNA was synthesized using extracted RNA by using cDNA primer: 5’- GTTTCCCAGTAGGTCTCNNNNNNNN-3’, and treated with Klenow DNA polymerase I (Promega) for end repair and PCR amplification was performed using the Roche Expand™ High Fidelity Plus kit (Sigma Aldrich, St. Louis, MO, USA) with primer: cgccGTTTCCCAGTAGGTCTC adapted from Palacios et al. [ ]. .. An Illumina Nextera XT DNA library was prepared from purified PCR products following manufacturer’s instructions.

Article Title: Microtubule nucleation promoters Mto1 and Mto2 regulate cytokinesis in fission yeast
Article Snippet: First, pFA6a-mto2S338N -C-KanMX6 was generated. .. A 245-base fragment of the end of the mto2 coding sequence was amplified from cps1-191 (containing the mto2S338N mutation) with flanking homology to the pFA6a vector using the Expand High Fidelity PCR system (Millipore Sigma). .. The vector backbone was amplified from pFA6a-GFP-KanMX6 using Phusion polymerase (New England BioLabs).

Synthesized:

Article Title: In Vitro and In Vivo Characterization of a Pigeon Paramyxovirus Type 1 Isolated from Domestic Pigeons in Victoria, Australia 2011
Article Snippet: .. Whole genome sequencing was performed using the Illumina MiSeq platform (Illumina, San Diego, CA, USA). cDNA was synthesized using extracted RNA by using cDNA primer: 5’- GTTTCCCAGTAGGTCTCNNNNNNNN-3’, and treated with Klenow DNA polymerase I (Promega) for end repair and PCR amplification was performed using the Roche Expand™ High Fidelity Plus kit (Sigma Aldrich, St. Louis, MO, USA) with primer: cgccGTTTCCCAGTAGGTCTC adapted from Palacios et al. [ ]. .. An Illumina Nextera XT DNA library was prepared from purified PCR products following manufacturer’s instructions.

Mutagenesis:

Article Title: Microtubule nucleation promoters Mto1 and Mto2 regulate cytokinesis in fission yeast
Article Snippet: First, pFA6a-mto2S338N -C-KanMX6 was generated. .. A 245-base fragment of the end of the mto2 coding sequence was amplified from cps1-191 (containing the mto2S338N mutation) with flanking homology to the pFA6a vector using the Expand High Fidelity PCR system (Millipore Sigma). .. The vector backbone was amplified from pFA6a-GFP-KanMX6 using Phusion polymerase (New England BioLabs).

Plasmid Preparation:

Article Title: Microtubule nucleation promoters Mto1 and Mto2 regulate cytokinesis in fission yeast
Article Snippet: First, pFA6a-mto2S338N -C-KanMX6 was generated. .. A 245-base fragment of the end of the mto2 coding sequence was amplified from cps1-191 (containing the mto2S338N mutation) with flanking homology to the pFA6a vector using the Expand High Fidelity PCR system (Millipore Sigma). .. The vector backbone was amplified from pFA6a-GFP-KanMX6 using Phusion polymerase (New England BioLabs).

Generated:

Article Title: sRNA Target Prediction Organizing Tool (SPOT) Integrates Computational and Experimental Data To Facilitate Functional Characterization of Bacterial Small RNAs
Article Snippet: Chromosomal mutations were made by λ Red recombination ( , ), and marked alleles were moved between strains by P1 vir transduction ( ). .. PCR products were generated using the Expand high-fidelity PCR system (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s instructions. .. All mutations were verified by amplifying PCR fragments using GoTaq polymerase (Promega, Madison, WI) and sequencing.

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    Millipore expand high fidelity pcr system
    Mechanisms of <t>GILZ</t> downregulation upon TLR activation. (A,B) AMs pretreated with BAY-11-7082 (BAY82, 5 μM), BAY-11-7085 (BAY85, 5 μM), or the solvent control DMSO (0.1%) for 1 h, followed by treatment with LPS (100 ng/mL), Pam 3 CSK 4 (Pam, 1 μg/mL), Poly(I:C) (PIC, 10 μg/mL), or medium (Co) for 4 h. GILZ expression was determined by Western blot. Tubulin served as a loading control. (A) Representative blot. (B) GILZ signal intensities were quantified and normalized to tubulin values ( n = 7). Values for unstimulated DMSO controls were set as 100%. (C,D) After preincubation with BAY-11-7082 or BAY-11-7085 (5 μM, 1 h), solvent (0.1% DMSO) or medium only (Co), AMs were treated with LPS (100 ng/mL) for 2 h. GILZ and ZFP36 mRNA expression was determined by <t>qRT-PCR</t> using ACTB as a housekeeping gene ( n = 3, duplicates). (E) AMs were either left untreated (Co) or treated with LPS (100 ng/mL), Pam 3 CSK 4 (Pam, 1 μg/mL), or Poly(I:C) (PIC, 10 μg/mL) for 4 h. TTP levels were determined by Western blot using tubulin as a loading control. GILZ signal intensities were normalized to tubulin and are shown as a percentage of untreated cells ( n = 2, triplicates). (F) The number of miRNAs predicted to target GILZ, CXCL8, IL6 , and TNF was assessed via the microRNA Data Integration Portal (mirDIP, accession date 02/02/2018). (G) AMs were either left untreated (Co) or treated with Poly(I:C) (PIC, 10 μg/mL), aurintricarboxylic acid (ATA, 25 μM), or a combination of both for 8 h. GILZ expression was quantified by Western blot. Signal intensities were normalized to tubulin and expressed as a percentage of untreated cells ( n = 3). * p
    Expand High Fidelity Pcr System, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/expand high fidelity pcr system/product/Millipore
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    expand high fidelity pcr system - by Bioz Stars, 2021-06
    94/100 stars
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    Mechanisms of GILZ downregulation upon TLR activation. (A,B) AMs pretreated with BAY-11-7082 (BAY82, 5 μM), BAY-11-7085 (BAY85, 5 μM), or the solvent control DMSO (0.1%) for 1 h, followed by treatment with LPS (100 ng/mL), Pam 3 CSK 4 (Pam, 1 μg/mL), Poly(I:C) (PIC, 10 μg/mL), or medium (Co) for 4 h. GILZ expression was determined by Western blot. Tubulin served as a loading control. (A) Representative blot. (B) GILZ signal intensities were quantified and normalized to tubulin values ( n = 7). Values for unstimulated DMSO controls were set as 100%. (C,D) After preincubation with BAY-11-7082 or BAY-11-7085 (5 μM, 1 h), solvent (0.1% DMSO) or medium only (Co), AMs were treated with LPS (100 ng/mL) for 2 h. GILZ and ZFP36 mRNA expression was determined by qRT-PCR using ACTB as a housekeeping gene ( n = 3, duplicates). (E) AMs were either left untreated (Co) or treated with LPS (100 ng/mL), Pam 3 CSK 4 (Pam, 1 μg/mL), or Poly(I:C) (PIC, 10 μg/mL) for 4 h. TTP levels were determined by Western blot using tubulin as a loading control. GILZ signal intensities were normalized to tubulin and are shown as a percentage of untreated cells ( n = 2, triplicates). (F) The number of miRNAs predicted to target GILZ, CXCL8, IL6 , and TNF was assessed via the microRNA Data Integration Portal (mirDIP, accession date 02/02/2018). (G) AMs were either left untreated (Co) or treated with Poly(I:C) (PIC, 10 μg/mL), aurintricarboxylic acid (ATA, 25 μM), or a combination of both for 8 h. GILZ expression was quantified by Western blot. Signal intensities were normalized to tubulin and expressed as a percentage of untreated cells ( n = 3). * p

    Journal: Frontiers in Immunology

    Article Title: Amplified Host Defense by Toll-Like Receptor-Mediated Downregulation of the Glucocorticoid-Induced Leucine Zipper (GILZ) in Macrophages

    doi: 10.3389/fimmu.2018.03111

    Figure Lengend Snippet: Mechanisms of GILZ downregulation upon TLR activation. (A,B) AMs pretreated with BAY-11-7082 (BAY82, 5 μM), BAY-11-7085 (BAY85, 5 μM), or the solvent control DMSO (0.1%) for 1 h, followed by treatment with LPS (100 ng/mL), Pam 3 CSK 4 (Pam, 1 μg/mL), Poly(I:C) (PIC, 10 μg/mL), or medium (Co) for 4 h. GILZ expression was determined by Western blot. Tubulin served as a loading control. (A) Representative blot. (B) GILZ signal intensities were quantified and normalized to tubulin values ( n = 7). Values for unstimulated DMSO controls were set as 100%. (C,D) After preincubation with BAY-11-7082 or BAY-11-7085 (5 μM, 1 h), solvent (0.1% DMSO) or medium only (Co), AMs were treated with LPS (100 ng/mL) for 2 h. GILZ and ZFP36 mRNA expression was determined by qRT-PCR using ACTB as a housekeeping gene ( n = 3, duplicates). (E) AMs were either left untreated (Co) or treated with LPS (100 ng/mL), Pam 3 CSK 4 (Pam, 1 μg/mL), or Poly(I:C) (PIC, 10 μg/mL) for 4 h. TTP levels were determined by Western blot using tubulin as a loading control. GILZ signal intensities were normalized to tubulin and are shown as a percentage of untreated cells ( n = 2, triplicates). (F) The number of miRNAs predicted to target GILZ, CXCL8, IL6 , and TNF was assessed via the microRNA Data Integration Portal (mirDIP, accession date 02/02/2018). (G) AMs were either left untreated (Co) or treated with Poly(I:C) (PIC, 10 μg/mL), aurintricarboxylic acid (ATA, 25 μM), or a combination of both for 8 h. GILZ expression was quantified by Western blot. Signal intensities were normalized to tubulin and expressed as a percentage of untreated cells ( n = 3). * p

    Article Snippet: Human GILZ 3′UTR cDNA was amplified using the Expand High fidelity PCR System (Sigma-Aldrich, # 11732641001) and the following primers: 5′-GCC TAC TAG TGC AGA GCC ACT AAA CTT G-3′ and 5′-AAT AGA GCT CAC TCT CAC AAA ACC CGC TAC-3′.

    Techniques: Activation Assay, Affinity Magnetic Separation, Expressing, Western Blot, Quantitative RT-PCR

    Mechanisms of GILZ downregulation upon TLR activation. (A,B) AMs pretreated with BAY-11-7082 (BAY82, 5 μM), BAY-11-7085 (BAY85, 5 μM), or the solvent control DMSO (0.1%) for 1 h, followed by treatment with LPS (100 ng/mL), Pam 3 CSK 4 (Pam, 1 μg/mL), Poly(I:C) (PIC, 10 μg/mL), or medium (Co) for 4 h. GILZ expression was determined by Western blot. Tubulin served as a loading control. (A) Representative blot. (B) GILZ signal intensities were quantified and normalized to tubulin values ( n = 7). Values for unstimulated DMSO controls were set as 100%. (C,D) After preincubation with BAY-11-7082 or BAY-11-7085 (5 μM, 1 h), solvent (0.1% DMSO) or medium only (Co), AMs were treated with LPS (100 ng/mL) for 2 h. GILZ and ZFP36 mRNA expression was determined by qRT-PCR using ACTB as a housekeeping gene ( n = 3, duplicates). (E) AMs were either left untreated (Co) or treated with LPS (100 ng/mL), Pam 3 CSK 4 (Pam, 1 μg/mL), or Poly(I:C) (PIC, 10 μg/mL) for 4 h. TTP levels were determined by Western blot using tubulin as a loading control. GILZ signal intensities were normalized to tubulin and are shown as a percentage of untreated cells ( n = 2, triplicates). (F) The number of miRNAs predicted to target GILZ, CXCL8, IL6 , and TNF was assessed via the microRNA Data Integration Portal (mirDIP, accession date 02/02/2018). (G) AMs were either left untreated (Co) or treated with Poly(I:C) (PIC, 10 μg/mL), aurintricarboxylic acid (ATA, 25 μM), or a combination of both for 8 h. GILZ expression was quantified by Western blot. Signal intensities were normalized to tubulin and expressed as a percentage of untreated cells ( n = 3). * p

    Journal: Frontiers in Immunology

    Article Title: Amplified Host Defense by Toll-Like Receptor-Mediated Downregulation of the Glucocorticoid-Induced Leucine Zipper (GILZ) in Macrophages

    doi: 10.3389/fimmu.2018.03111

    Figure Lengend Snippet: Mechanisms of GILZ downregulation upon TLR activation. (A,B) AMs pretreated with BAY-11-7082 (BAY82, 5 μM), BAY-11-7085 (BAY85, 5 μM), or the solvent control DMSO (0.1%) for 1 h, followed by treatment with LPS (100 ng/mL), Pam 3 CSK 4 (Pam, 1 μg/mL), Poly(I:C) (PIC, 10 μg/mL), or medium (Co) for 4 h. GILZ expression was determined by Western blot. Tubulin served as a loading control. (A) Representative blot. (B) GILZ signal intensities were quantified and normalized to tubulin values ( n = 7). Values for unstimulated DMSO controls were set as 100%. (C,D) After preincubation with BAY-11-7082 or BAY-11-7085 (5 μM, 1 h), solvent (0.1% DMSO) or medium only (Co), AMs were treated with LPS (100 ng/mL) for 2 h. GILZ and ZFP36 mRNA expression was determined by qRT-PCR using ACTB as a housekeeping gene ( n = 3, duplicates). (E) AMs were either left untreated (Co) or treated with LPS (100 ng/mL), Pam 3 CSK 4 (Pam, 1 μg/mL), or Poly(I:C) (PIC, 10 μg/mL) for 4 h. TTP levels were determined by Western blot using tubulin as a loading control. GILZ signal intensities were normalized to tubulin and are shown as a percentage of untreated cells ( n = 2, triplicates). (F) The number of miRNAs predicted to target GILZ, CXCL8, IL6 , and TNF was assessed via the microRNA Data Integration Portal (mirDIP, accession date 02/02/2018). (G) AMs were either left untreated (Co) or treated with Poly(I:C) (PIC, 10 μg/mL), aurintricarboxylic acid (ATA, 25 μM), or a combination of both for 8 h. GILZ expression was quantified by Western blot. Signal intensities were normalized to tubulin and expressed as a percentage of untreated cells ( n = 3). * p

    Article Snippet: Human GILZ 3′UTR cDNA was amplified using the Expand High fidelity PCR System (Sigma-Aldrich, # 11732641001) and the following primers: 5′-GCC TAC TAG TGC AGA GCC ACT AAA CTT G-3′ and 5′-AAT AGA GCT CAC TCT CAC AAA ACC CGC TAC-3′.

    Techniques: Activation Assay, Affinity Magnetic Separation, Expressing, Western Blot, Quantitative RT-PCR