Journal: Cell Reports Medicine

Article Title: A glucocorticoid spike derails muscle repair to heterotopic ossification after spinal cord injury

doi: 10.1016/j.xcrm.2024.101849

Figure Lengend Snippet:

Article Snippet: As mentioned above, leukocytes were isolated from mouse hamstring muscles 4 days post-injury using a skeletal muscle dissociation kit (Miltenyi Biotech) as per manufactures instructions.

Techniques: Control, Recombinant, Plasmid Preparation, Staining, cDNA Synthesis, Concentration Assay, Bicinchoninic Acid Protein Assay, RNA Sequencing, Saline, Gene Expression, Microarray, Software, Western Blot, Imaging, Cytometry, Tomography

Journal: Scientific Reports

Article Title: Expression profiling of prostate cancer tissue delineates genes associated with recurrence after prostatectomy

doi: 10.1038/srep16018

Figure Lengend Snippet: ( A ) Example of each of the staining intensities 0-to 3 ( B ) Kaplan-Meier plot of recurrence free survival as a function of SFRP4 protein expression. ( C ) Correlation plot between the intensity of SFRP4 measured by IHC and the expression level of SFRP4 measured by microarray analysis from the same patients. ( D ) Correlation plot between SFRP4 gene expression measured by Affymetrix array and by q-RTPCR.

Article Snippet: A Taq-man primer set targeting SFRP4 (assay id Hs00180066 m1*) was used for the real-time PCR and UBC was included for normalization, and measured using primers 5′-GATTTGGGTCGCGGTTCTT-3′ plus 5′-TGCCTTGACATTCTCGATGGT-3′ and SYBR Green PCR Master Mix (Applied Biosystems) .

Techniques: Staining, Expressing, Microarray, Gene Expression, Reverse Transcription Polymerase Chain Reaction

Journal:

Article Title: Dominant Activation of the Hedgehog Signaling Pathway in the Ovary Alters Theca Development and Prevents Ovulation

doi: 10.1210/me.2008-0391

Figure Lengend Snippet: Results of microarray analysis of ovarian RNA from Amhr2 cre /+ SmoM2 (mutant) and Amhr2 +/+ SmoM2 (control) mice, and confirmation by quantitative RT-PCR

Article Snippet: The between-plate coefficients of variation ranged from 1% to 16%; the mean was 10.6 ± 1.4%. table ft1 table-wrap mode="anchored" t5 Table 2 caption a7 Gene symbol Gene name Assay identification a Exons b Smo Smoothened homolog Mm01162710_m1 8–9 Ihh Indian hedgehog Mm00439613_m1 2–3 Ptch1 Patched homolog 1 Mm00436026_m1 17–18 Gli1 GLI-Kruppel family member GLI1 Mm00494645_m1 2–3 Gli3 GLI-Kruppel family member GLI3 Mm00492333_m1 1–2 Hhip Hedgehog-interacting protein Mm00469580_m1 12–13 Spp Secreted phosphoprotein 1 Mm00436767_m1 2–3 Ptgs2 Prostaglandin-endoperoxidase synthase 2 Mm00478374_m1 5–6 Pgr Progesterone receptor Mm00435625_m1 4–5 Star Steroidogenic acute regulatory protein Mm00441558_m1 6–7 Lhcgr LH/CG receptor Mm00442931_m1 6–7 Actg2 Actin, γ 2, smooth muscle, enteric Mm00656102_m1 7–8 Cnn1 Calponin 1 Mm00487032_m1 1–2 Des Desmin Mm00802455_m1 1–2 Tagln Transgelin Mm00441660_m1 1–2 Tnc Tenascin C Mm00495662_m1 5–6 Edn2 Endothelin 2 Mm00432983_m1 3–4 18S rRNA 4319413E Open in a separate window a Taqman Gene Expression Assays (Applied Biosystems). b Exons in which forward and reverse primers anneal.

Techniques: Microarray, Mutagenesis, Control, Quantitative RT-PCR, Sequencing

Journal:

Article Title: Dominant Activation of the Hedgehog Signaling Pathway in the Ovary Alters Theca Development and Prevents Ovulation

doi: 10.1210/me.2008-0391

Figure Lengend Snippet: Expression of genes associated with smooth muscle (Cnn1, Des, Actg2, Tagln) or extracellular matrix (Tnc) and expression of Edn2 in granulosa cells and residual ovarian tissue from Amhr2+/+SmoM2 and Amhr2cre/+SmoM2 mice. Tissues were obtained from untreated mice (UT) or from eCG-primed mice before (0 h) or after injection of hCG. Total RNA was assayed by quantitative real-time RT-PCR. Data are mean ± sem; n = 3 granulosa cell and residual tissue preparations. Bars without common superscripts are significantly different (P < 0.05).

Article Snippet: The between-plate coefficients of variation ranged from 1% to 16%; the mean was 10.6 ± 1.4%. table ft1 table-wrap mode="anchored" t5 Table 2 caption a7 Gene symbol Gene name Assay identification a Exons b Smo Smoothened homolog Mm01162710_m1 8–9 Ihh Indian hedgehog Mm00439613_m1 2–3 Ptch1 Patched homolog 1 Mm00436026_m1 17–18 Gli1 GLI-Kruppel family member GLI1 Mm00494645_m1 2–3 Gli3 GLI-Kruppel family member GLI3 Mm00492333_m1 1–2 Hhip Hedgehog-interacting protein Mm00469580_m1 12–13 Spp Secreted phosphoprotein 1 Mm00436767_m1 2–3 Ptgs2 Prostaglandin-endoperoxidase synthase 2 Mm00478374_m1 5–6 Pgr Progesterone receptor Mm00435625_m1 4–5 Star Steroidogenic acute regulatory protein Mm00441558_m1 6–7 Lhcgr LH/CG receptor Mm00442931_m1 6–7 Actg2 Actin, γ 2, smooth muscle, enteric Mm00656102_m1 7–8 Cnn1 Calponin 1 Mm00487032_m1 1–2 Des Desmin Mm00802455_m1 1–2 Tagln Transgelin Mm00441660_m1 1–2 Tnc Tenascin C Mm00495662_m1 5–6 Edn2 Endothelin 2 Mm00432983_m1 3–4 18S rRNA 4319413E Open in a separate window a Taqman Gene Expression Assays (Applied Biosystems). b Exons in which forward and reverse primers anneal.

Techniques: Expressing, Injection, Quantitative RT-PCR

Journal:

Article Title: Dominant Activation of the Hedgehog Signaling Pathway in the Ovary Alters Theca Development and Prevents Ovulation

doi: 10.1210/me.2008-0391

Figure Lengend Snippet: Quantitative real-time RT-PCR assays

Article Snippet: The between-plate coefficients of variation ranged from 1% to 16%; the mean was 10.6 ± 1.4%. table ft1 table-wrap mode="anchored" t5 Table 2 caption a7 Gene symbol Gene name Assay identification a Exons b Smo Smoothened homolog Mm01162710_m1 8–9 Ihh Indian hedgehog Mm00439613_m1 2–3 Ptch1 Patched homolog 1 Mm00436026_m1 17–18 Gli1 GLI-Kruppel family member GLI1 Mm00494645_m1 2–3 Gli3 GLI-Kruppel family member GLI3 Mm00492333_m1 1–2 Hhip Hedgehog-interacting protein Mm00469580_m1 12–13 Spp Secreted phosphoprotein 1 Mm00436767_m1 2–3 Ptgs2 Prostaglandin-endoperoxidase synthase 2 Mm00478374_m1 5–6 Pgr Progesterone receptor Mm00435625_m1 4–5 Star Steroidogenic acute regulatory protein Mm00441558_m1 6–7 Lhcgr LH/CG receptor Mm00442931_m1 6–7 Actg2 Actin, γ 2, smooth muscle, enteric Mm00656102_m1 7–8 Cnn1 Calponin 1 Mm00487032_m1 1–2 Des Desmin Mm00802455_m1 1–2 Tagln Transgelin Mm00441660_m1 1–2 Tnc Tenascin C Mm00495662_m1 5–6 Edn2 Endothelin 2 Mm00432983_m1 3–4 18S rRNA 4319413E Open in a separate window a Taqman Gene Expression Assays (Applied Biosystems). b Exons in which forward and reverse primers anneal.

Techniques: Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Comparative transcriptome analysis of inner blood-retinal barrier and blood–brain barrier in rats

doi: 10.1038/s41598-021-91584-7

Figure Lengend Snippet: Features of the assays used for semiquantitative real time polymerase chain reaction (qRT-PCR) for validation and conformation of microarray measurements.

Article Snippet: Rattus norvegicus ATP binding cassette subfamily C member 4 , Abcc4 , NM_133411.1 , Rn01465702_m1 , 914 , 64.

Techniques: Real-time Polymerase Chain Reaction, Biomarker Discovery, Microarray, Amplification, Binding Assay, Derivative Assay

Journal: Scientific Reports

Article Title: Comparative transcriptome analysis of inner blood-retinal barrier and blood–brain barrier in rats

doi: 10.1038/s41598-021-91584-7

Figure Lengend Snippet: Enrichment of 6 target genes in retina microvessels (RMVs) and brain microvessels (BMVs) over the respective full tissues. (A) Fold changes obtained in the semiquantitative RT-PCR (qRT-PCR) measurements in RMVs (open bars) and BMVs (filled bars). Gja5, gap junction alpha-5 protein (also termed Cx40); Slc2A1, solute carrier family 2 member 1; Slc7A1, solute carrier family 7 member 1; Slc38A5, solute carrier family 38 member 5; Abcc4, ATP binding cassette subfamily C member 4; Pdgfrb, platelet derived growth factor receptor beta. (B) Agreement of results obtained by microarray measurements (abscissa) and qRT-PCR measurements (ordinate). In this graph enrichment is indicated as log 2 fold changes. The regression line and the corresponding correlation coefficient show the significant agreement of data obtained by the two methods.

Article Snippet: Rattus norvegicus ATP binding cassette subfamily C member 4 , Abcc4 , NM_133411.1 , Rn01465702_m1 , 914 , 64.

Techniques: Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Binding Assay, Derivative Assay, Microarray

Journal: Scientific Reports

Article Title: Comparative transcriptome analysis of inner blood-retinal barrier and blood–brain barrier in rats

doi: 10.1038/s41598-021-91584-7

Figure Lengend Snippet: List of ATP binding cassette transporter genes differentially expressed in rat retina and/or brain microvessels vs. retina and brain tissue samples.

Article Snippet: Rattus norvegicus ATP binding cassette subfamily C member 4 , Abcc4 , NM_133411.1 , Rn01465702_m1 , 914 , 64.

Techniques: Binding Assay

Journal: Cell reports

Article Title: Mesothelial Cell HIF1α Expression Is Metabolically Downregulated by Metformin to Prevent Oncogenic Tumor-Stromal Crosstalk

doi: 10.1016/j.celrep.2019.11.079

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Recombinant DNA HA-HIF1α P402A/P564A-pcDNA3 Yan et al., 2007 Addgene: 338299 pH IV-ODD-EGFP-IRES-dTomato Bagnall et al., 2014 Addgene: 395015 SUCLG2 DNASU HsCD00445557 CXCR1 Sino Biological HG10856-NF CCR2 Sino Biological HG16084-NF EF1a-GFP-IRES-Puro (pEGIP) Zou et al., 2009 Addgene: 26777 Software and Algorithms GraphPad Prism 7 GraphPad Software RRID: SCR_002798 FIJI [ImageJ] National Institutes of Health RRID:SCR_002285 Aperio ScanScope / Pixel Count v9 Leica Biosystems RRID: SCR_014311 Thermo Xcalibur ThermoFisher Scientific RRID: SCR_014593 MaxQuant Max Planck Institute RRID: SCR_014485 Perseus Max Planck Institute RRID: SCR_015753 Molecular Signatures Database Broad Institute, MIT RRID: SCR_016863 MetaboAnalyst McGill University RRID: SCR_015539 Other Invasion transwell insert (8μm pore) ThermoFisher Scientific 353097 Co-culture transwell insert (0.4μm pore) Thomas Scientific 3450 Collagen type 1 [rat tail] Corning 354236 PureLink HiPure Plasmid Maxiprep Kit ThermoFisher Scientific K2100-06 IDEXX CellCheck IDEXX Bioresearch https://www.idexxbioanalytics.com/cellcheck Open in a separate window KEY RESOURCES TABLE

Techniques: Ex Vivo, Isolation, Microarray, Recombinant, Mutagenesis, Enzyme-linked Immunosorbent Assay, Software, Plasmid Preparation

Journal: Cell reports

Article Title: Cell-Autonomous Regulation of Astrocyte Activation by the Circadian Clock Protein BMAL1

doi: 10.1016/j.celrep.2018.09.015

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Gsta4 Taqman Gene expression assay , Applied Biosystems , Mm00494803_m1.

Techniques: Virus, Plasmid Preparation, Recombinant, Transfection, Detection Assay, Microarray, Mutagenesis, Control, Gene Expression, Software

Journal: Nature Communications

Article Title: MRF4 negatively regulates adult skeletal muscle growth by repressing MEF2 activity

doi: 10.1038/ncomms12397

Figure Lengend Snippet: ( a ) Rat soleus muscles co-transfected with GFP and LacZ or Mrf4 shRNAs and examined 14 days later. Scale bar, 100 μm. Cross-sectional area (CSA) of transfected fibres is shown below. Two hundred GFP-positive fibres from each muscle were analysed. ( b ) CSA of muscle fibres in innervated or 7 d denervated SOL and EDL muscles transfected with two different Mrf4 shRNAs (M1 and M2). Values normalized to fibres transfected with LacZ shRNAs in innervated muscles. Measures on 58 muscles, about 5 muscles per group, 15,485 fibres. ( c ) Mrf4 overexpression in adult SOL. CSA of muscle fibres transfected with Mrf4 cDNA normalized to control muscles ( n =3). ( d ) Fibre size is increased in regenerating SOL co-transfected at day 3 after injury with GFP and LacZ or Mrf4 shRNAs, and examined 7 days later. Scale bar 100 μm. Right: CSA of regenerating fibres transfected with Mrf4 shRNAs (M1) normalized to control ( n =3). ( e ) Fibre size is reduced by Mrf4 overexpression in regenerating muscle ( n =3). ( f ) Rescue experiment for M1. The sequence recognized by M1 shRNA in rat Mrf4 transcripts (rMrf4) has a single base difference in human Mrf4 (hMrf4). hMrf4 is not silenced by M1 in HEK-293 cells (left), as shown by western blotting with anti-MRF4. Fibre growth induced by M1 in 14 days denervated SOL is prevented by M1-resistant hMrf4 (right, n =5). ( g ) Rescue experiment for M2. Same as in f, but using M2 shRNA specific for rat Mrf4 and M2-resistant mouse Mrf4 (mMrf4) ( n =5). ( h ) Fibre size is not affected by myogenin knockdown. Left: HEK-293 cells transfected with myogenin cDNA (MyoG) and co-transfected with two shRNAs targeting myogenin (MG1 and MG2). GFP was co-transfected to determine transfection efficiency. Right: fibre size in unchanged by MG1 ( n =3). ( i ) Increased Protein synthesis is increased in SOL by Mrf4 shRNAs (M1), as revealed by puromycin incorporation and immunostaining for puromycin-labelled peptides. Quantification is on the right ( n =4). Data are presented as mean±s.e.m. from at least three independent experiments. Statistical analysis with Student's two-tailed t -test (* P <0.05, ** P <0.01, *** P <0.001).

Article Snippet: Rat Mrf4 and MyoG were from Addgene; human Mrf4 from Origene.

Techniques: Muscles, Transfection, Over Expression, Control, Sequencing, shRNA, Western Blot, Knockdown, Immunostaining, Two Tailed Test

Journal: Nature Communications

Article Title: MRF4 negatively regulates adult skeletal muscle growth by repressing MEF2 activity

doi: 10.1038/ncomms12397

Figure Lengend Snippet: ( a ) Heatmap representing the top-ranking 100 genes showing differential expression between M1 and control. Each lane corresponds to one animal, three animals per group were examined. Note that innervated (INN) and denervated (DEN) M1 groups display similar changes in gene expression and that genes upregulated by M1 are predominant in this heatmap. ( b ) Gene Set Enrichment Analysis (GSEA) reporting the groups of genes of the GO Cellular Component positively enriched in muscles transfected with shRNAs against Mrf4 (M1 sequence). NES: Normalized Enrichment Score; FDR: false discovery rate. ( c ) Myosin heavy and light chains upregulated by Mrf4 knockdown ( P <0.05; fold change≥1.5). ( d ) Increased levels of myosin heavy chain (MyHC) transcripts coded by Myh2 and Myh6 in muscles transfected with Mrf4 RNAi (M1) compared to control LacZ RNAi, as determined by qPCR ( n =3, same samples used for microarray analysis in a ). Data are presented as means±s.e.m. Statistical analysis was performed using Student's two-tailed t -test, ** P <0.01. ( e ) SOL muscle transfected with Mrf4 shRNA and examined at 14 days after transfection. Serial sections stained with anti-MyHC-2A ( Myh2 ) and MyHC-α cardiac ( Myh6 ) reveal that a number of transfected fibres staining for MyHC-2A are also reactive for MyHC-α cardiac. Scale bar, 100 μm.

Article Snippet: Rat Mrf4 and MyoG were from Addgene; human Mrf4 from Origene.

Techniques: Quantitative Proteomics, Control, Gene Expression, Muscles, Transfection, Sequencing, Knockdown, Microarray, Two Tailed Test, shRNA, Staining

Journal: Nature Communications

Article Title: MRF4 negatively regulates adult skeletal muscle growth by repressing MEF2 activity

doi: 10.1038/ncomms12397

Figure Lengend Snippet: ( a ) Changes in expression of transcripts coding for myogenin, MyoD, MEF2a, Mef2c and Mef2d induced by MRF4 knockdown, as determined by qPCR ( n =3, same samples used for microarray analysis in ). ( b ) Expression pattern of MEF2 target genes, as determined by crossing the results obtained from GSEA (TRANSFAC) and differential expression analysis. Red and green colours represent relative gene expression levels (red, maximum; green, minimum). ( c ) qPCR shows increased levels of MEF2 targets, including the genes coding for the actin-binding Rho-activating protein ( Abra ), also called STARS, creatine kinase ( Ckm ) and the glucose transporter Glut4, in muscles transfected with Mrf4 RNAi (M1) compared with control LacZ RNAi ( n =3, same samples used for microarray analysis in ). qPCR data are presented as means±s.e.m. Statistical analysis was performed using Student's two-tailed t -test (* P <0.05, ** P <0.01, *** P <0.001).

Article Snippet: Rat Mrf4 and MyoG were from Addgene; human Mrf4 from Origene.

Techniques: Expressing, Knockdown, Microarray, Quantitative Proteomics, Gene Expression, Binding Assay, Muscles, Transfection, Control, Two Tailed Test

Journal: Nature Communications

Article Title: MRF4 negatively regulates adult skeletal muscle growth by repressing MEF2 activity

doi: 10.1038/ncomms12397

Figure Lengend Snippet: ( a ) HEK-293 cells were transfected with MEF2C-Flag and co-transfected with myc-tagged MRF4 or myc-tagged myogenin (MyoG). Cell lysates were immunoprecipitated with an anti-Flag antibody and resolved by SDS–PAGE, followed by western blotting with anti-Myc antibody. MRF4, like MyoG, is co-immunoprecipitated with MEF2, and MEF2 is co-immunoprecipitated with MRF4 or MyoG. ( b ) HEK-293 cells co-transfected with MEF2C, a MEF2-Luciferase reporter, containing three tandem copies of the MEF2 site linked to luciferase, and either MyoD, myogenin or MRF4 ( n =3). ( c ) Adult SOL muscle transfected with a MEF2-Luciferase reporter were co-transfected with Mrf4 or myogenin ( n =4). ( d ) Adult SOL muscle transfected with a MEF2-Luciferase reporter were co-transfected with Mrf4 RNAi (M1) or myogenin RNAi (MG1) ( n =4). ( e ) Adult SOL muscle transfected with a MEF2-Luciferase reporter were co-transfected with Mrf4 RNAi (M2) in the presence or absence of the M2-resistant mouse Mrf4 (mMrf4) ( n =4). ( f ) Structure of two MRF4-myogenin chimeras containing either the N-terminal domain of MRF4 (MR) and the C-terminal domain of myogenin (MG) (chimera 1), or the N-terminal domain of myogenin and the C-terminal domain of MRF4 (chimera 2). ( g ) SOL and EDL muscles were transfected with the MEF2 reporter and co-transfected with either myogenin, Mrf4 or chimeric transgenes ( n =5). Data are presented as means±s.e.m. from at least three independent experiments. Statistical analysis was performed using Student's two-tailed t -test (* P <0.05, ** P <0.01, *** P <0.001).

Article Snippet: Rat Mrf4 and MyoG were from Addgene; human Mrf4 from Origene.

Techniques: Transfection, Immunoprecipitation, SDS Page, Western Blot, Luciferase, Muscles, Two Tailed Test

Journal: Nature Communications

Article Title: MRF4 negatively regulates adult skeletal muscle growth by repressing MEF2 activity

doi: 10.1038/ncomms12397

Figure Lengend Snippet: ( a ) Scheme of the MEF2 constructs used for transfection experiments. ( b ) Innervated or denervated adult SOL muscles were transfected with Mrf4 shRNA (M1), dominant negative MEF2 (dnMEF2), or co-transfected with both constructs, compared to control muscles ( n =3). ( c ) Denervated SOL muscle transfected with a constitutively active and inducible MEF2 construct (MEF2-VP16-ER) and co-transfected with GFP. Rats were treated with tamoxifen every other day for 7 days. Transverse sections from denervated transfected SOL stained for dystrophin and co-stained for GFP (left panel) or, on a serial section, for VP16 (central panel) or VP16 and GFP (right panel). Scale bar, 50 μm. ( d ) Innervated and denervated SOL muscles were transfected with caMEF2 or with DNA-binding-deficient Δ-caMEF2. Rats were treated with tamoxifen every other day for 7 days. Cross-sectional areas of transfected fibres normalized to untransfected fibres ( n =4). ( e ) Innervated SOL muscle were transfected with caMEF2, but rats were treated with oil vehicle rather than tamoxifen. Transverse section was stained for VP16. Scale bar, 50 μm. ( f ) Innervated SOL muscles were transfected with either caMEF2 or Δ-caMEF2, rats were treated with oil vehicle. Cross-sectional areas of transfected fibres normalized to untransfected fibres ( n =5). Data are presented as means±s.e.m. from at least three independent experiments. Statistical analysis was performed using Student's two-tailed t -test (* P <0.05, ** P <0.01, *** P <0.001).

Article Snippet: Rat Mrf4 and MyoG were from Addgene; human Mrf4 from Origene.

Techniques: Construct, Transfection, Muscles, shRNA, Dominant Negative Mutation, Control, Staining, Binding Assay, Two Tailed Test

Journal: Nature Communications

Article Title: MRF4 negatively regulates adult skeletal muscle growth by repressing MEF2 activity

doi: 10.1038/ncomms12397

Figure Lengend Snippet: ( a , b ) HDAC4 distribution in innervated ( a ) and denervated ( b ) SOL muscle. Sections stained for HDAC4 (red) and dystrophin (green). Note nuclear translocation of HDAC4 into myonuclei induced by denervation. Scale bar, 50 μm. ( c ) Denervated SOL muscle transfected with Mrf4 shRNAs (M1) and co-transfected with GFP. Serial sections were stained for dystrophin and co-stained for GFP (left panel) or for HDAC4 (right panel). Note impaired nuclear accumulation of HDAC4 into transfected hypertrophic myofibres. Four transfected fibres are labelled with asterisks. Scale bar, 50 μm. ( d ) Quantitative analysis of percentage nuclear distribution of HDAC4 in innervated and denervated SOL muscles transfected with shRNAs against Mrf4 or LacZ ( n =3). ( e ) Western blot of HDAC4 expression in innervated and denervated SOL muscles transfected with Mrf4 (M1) or LacZ shRNAs. Protein content obtained from band quantification normalized to innervated (INN) control muscles is below ( n =3). HDAC4 is about 5-fold more abundant in denervated compared to innervated muscles, but values are similar in LacZ and M1. Data are presented as means±s.e.m. from at least three independent experiments. Statistical analysis was performed using Student's two-tailed t -test (* P <0.05, ** P <0.01, *** P <0.001).

Article Snippet: Rat Mrf4 and MyoG were from Addgene; human Mrf4 from Origene.

Techniques: Staining, Translocation Assay, Transfection, Muscles, Western Blot, Expressing, Control, Two Tailed Test

Journal: Journal of Cellular and Molecular Medicine

Article Title: Machine learning approach identifies new pathways associated with demyelination in a viral model of multiple sclerosis

doi: 10.1111/j.1582-4934.2008.00646.x

Figure Lengend Snippet: Temporal changes of leucocytic infiltrates and demyelination in the spinal cord. Box and whiskerplots show the median and quartiles of the density of (A) CD3, (B) CD45R/B220, (C) IgG, (D) CD107b + cells and (E) the MBP + area. Extreme values are shown as circles. Six mice were evaluated per group and time-point, except for five TMEV-infected mice at 98 dpi. A significant difference between the groups as detected by the Mann-Whitney U-test is marked as follows: * P ≤ 0.05.

Article Snippet: The applied antibodies were CD3 (polyclonal rabbit anti-human, diluted 1: 1000, Dako Diagnostika, Hamburg, Germany), CD45R/B220 (monoclonal rat antimouse biotinylated, clone RA3–6B2, diluted 1: 2000, BD Biosciences Pharmingen, Erembodegem, Belgium), IgG (H + L, polyclonal goat antimouse biotinylated, diluted 1: 200, Biologo, Kiel Germany), CD107b (monoclonal rat antimouse biotinylated, clone M/4, diluted 1: 800, Serotec, Oxford, UK) and myelin basic protein (MBP; polyclonal rabbit anti-human, diluted 1: 800, Chemicon Europe, Hofheim/Taunus, Germany).

Techniques: Infection, MANN-WHITNEY

Journal: Journal of Cellular and Molecular Medicine

Article Title: Machine learning approach identifies new pathways associated with demyelination in a viral model of multiple sclerosis

doi: 10.1111/j.1582-4934.2008.00646.x

Figure Lengend Snippet: Immunohistological characterization of a demyelinating lesion at 98 dpi. Serial sections from the spinal cord of a TMEV-infected mouse from 98 dpi were immunolabelled for CD3 (A), CD45R/B220 (B), IgG (C), CD107b (D) and MBP (E). (A) A high amount of CD3 + T lymphocytes in the perivascular spaces and fewer cells in the white matter (arrows). (B) Few perivascular CD45R/B220 + B lymphocytes (arrow). (C) A high amount of IgG + cells in the perivascular spaces (arrown). (D) CD107b + macrophages with Gitter cell morphology (arrows) comprise the majority of parenchymal cellular infiltrates (E) Multifocally decreased MBP immunoreactivity (arrow) in the white matter. m = meninges, v = vessel, wm = white matter. Avidin-biotin-peroxidase complex method, haematoxylin counter-stain, Nomarski differential interference contrast. Scale bars = 20 μm.

Article Snippet: The applied antibodies were CD3 (polyclonal rabbit anti-human, diluted 1: 1000, Dako Diagnostika, Hamburg, Germany), CD45R/B220 (monoclonal rat antimouse biotinylated, clone RA3–6B2, diluted 1: 2000, BD Biosciences Pharmingen, Erembodegem, Belgium), IgG (H + L, polyclonal goat antimouse biotinylated, diluted 1: 200, Biologo, Kiel Germany), CD107b (monoclonal rat antimouse biotinylated, clone M/4, diluted 1: 800, Serotec, Oxford, UK) and myelin basic protein (MBP; polyclonal rabbit anti-human, diluted 1: 800, Chemicon Europe, Hofheim/Taunus, Germany).

Techniques: Infection, Avidin-Biotin Assay, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Machine learning approach identifies new pathways associated with demyelination in a viral model of multiple sclerosis

doi: 10.1111/j.1582-4934.2008.00646.x

Figure Lengend Snippet: Correlation between immunohistology and gene expression as measured by microarray. Spearman’s rank correlation coefficient was used to correlate the density of CD3, CD45R/B220, IgG and CD107b + cells, and the myelin basic protein positive area with the respective mRNA level measured by microarray analysis in the spinal cord

Article Snippet: The applied antibodies were CD3 (polyclonal rabbit anti-human, diluted 1: 1000, Dako Diagnostika, Hamburg, Germany), CD45R/B220 (monoclonal rat antimouse biotinylated, clone RA3–6B2, diluted 1: 2000, BD Biosciences Pharmingen, Erembodegem, Belgium), IgG (H + L, polyclonal goat antimouse biotinylated, diluted 1: 200, Biologo, Kiel Germany), CD107b (monoclonal rat antimouse biotinylated, clone M/4, diluted 1: 800, Serotec, Oxford, UK) and myelin basic protein (MBP; polyclonal rabbit anti-human, diluted 1: 800, Chemicon Europe, Hofheim/Taunus, Germany).

Techniques: Gene Expression, Microarray, Expressing

Journal: Scientific Reports

Article Title: Assessing the contribution of thrombospondin-4 induction and ATF6α activation to endoplasmic reticulum expansion and phenotypic modulation in bladder outlet obstruction

doi: 10.1038/srep32449

Figure Lengend Snippet: Rats were either sham-operated or obstructed for 10 days and 6 weeks, respectively. In one group of rats the obstruction was relieved for 10 days following 6 weeks of obstruction. Analysis of gene activity by microarray analysis (GSE47080, n = 6–8 at all times) demonstrated sizeable induction of Thbs4 (panel a). Independent confirmation by qRT-PCR of the up-regulation of Thbs4 is shown in panel b. Panel c shows immunoblotting (IB) for the Thbs4 protein in control and obstructed bladders. Two different size standards were used. Panel d shows summarized data on the Thbs4 protein at different times of obstruction. Means ± S.E.M. of 6–8 independent replicates are depicted in this and the following figures.

Article Snippet: After protein separation and semi-dry transfer to nitrocellulose for 10–20 min in the Trans-Blot Turbo system, we cut the membranes horizontally to allow for blotting of multiple targets using the following antibodies: thrombospondin-4 (Thbs4, sc-7657-R and sc-390734, Santa Cruz Biotechnology, AF2390, R&D systems and PA5-34386, Thermo Scientific), ATF6α (Atf6, ab11909 (a.k.a.

Techniques: Activity Assay, Microarray, Quantitative RT-PCR, Western Blot

Journal: Scientific Reports

Article Title: Assessing the contribution of thrombospondin-4 induction and ATF6α activation to endoplasmic reticulum expansion and phenotypic modulation in bladder outlet obstruction

doi: 10.1038/srep32449

Figure Lengend Snippet: Rats were sham-operated or obstructed for 2, 4, and 10 days. Following bladder excision, the detrusor and mucosal layers were separated by microdissection and RNA was isolated. qRT-PCR showed significant Thbs4 induction in the detrusor ( a ) but no change in the mucosa ( b ). Immunohistochemistry (DAB, brown, panels c and d) for Thbs4 confirmed increased staining as well as thickening of the detrusor layer after 6 weeks of obstruction. Thbs4 positive cells are indicated by black arrowheads in panel d. Examination of detrusor at higher magnification using immunofluorescence revealed that Thbs4 (green) accumulated intracellularly close to the nuclear poles (panels e,f). Black and white scale bars represent 200 and 100 μm, respectively. The inset in ( f ) shows one example of a Thbs4 positive cell where Thbs4 staining (green) radiates out from the nucleus (blue).

Article Snippet: After protein separation and semi-dry transfer to nitrocellulose for 10–20 min in the Trans-Blot Turbo system, we cut the membranes horizontally to allow for blotting of multiple targets using the following antibodies: thrombospondin-4 (Thbs4, sc-7657-R and sc-390734, Santa Cruz Biotechnology, AF2390, R&D systems and PA5-34386, Thermo Scientific), ATF6α (Atf6, ab11909 (a.k.a.

Techniques: Laser Capture Microdissection, Isolation, Quantitative RT-PCR, Immunohistochemistry, Staining, Immunofluorescence

Journal: Scientific Reports

Article Title: Assessing the contribution of thrombospondin-4 induction and ATF6α activation to endoplasmic reticulum expansion and phenotypic modulation in bladder outlet obstruction

doi: 10.1038/srep32449

Figure Lengend Snippet: The 50 kDa cleaved and active form of ATF6α, spliced XBP1 (Xbp1s, 54 kDa) and phosphorylated Perk/eIF2α, three arms of the classical ER stress pathway, were assayed by western blotting at different times of outlet obstruction ( a ). Summarized data on ATF6α-P50 is shown in panel b and summarized data on the spliced form of XBP1 is shown in ( c ). Panel d shows time courses of ATF6α target genes from the microarray experiment (GSE47080, the P-value is for comparison of 10 d vs. sham). Panel e shows protein levels of ATF6α targets in a new experiment (different set of bladders than those in Fig. 3a). Panel f shows that Thbs4 mRNA is induced in the mouse bladder following 7 days of outlet obstruction. Panel g shows a stack of blots for Thbs4, ATF6α and ATF6α target genes in control and obstructed mouse bladders. Panel h and i show correlation analyses for Thbs4 versus cleaved (50 kDa) over total ATF6α and for cleaved over total ATF6α versus Pdi, respectively. P-values from Spearmann correlation analyses are given in the panels.

Article Snippet: After protein separation and semi-dry transfer to nitrocellulose for 10–20 min in the Trans-Blot Turbo system, we cut the membranes horizontally to allow for blotting of multiple targets using the following antibodies: thrombospondin-4 (Thbs4, sc-7657-R and sc-390734, Santa Cruz Biotechnology, AF2390, R&D systems and PA5-34386, Thermo Scientific), ATF6α (Atf6, ab11909 (a.k.a.

Techniques: Western Blot, Microarray

Journal: Scientific Reports

Article Title: Assessing the contribution of thrombospondin-4 induction and ATF6α activation to endoplasmic reticulum expansion and phenotypic modulation in bladder outlet obstruction

doi: 10.1038/srep32449

Figure Lengend Snippet: Bladder, aorta and ileum were harvested from C57BL/6J mice and divided into equal halves. One half was immediately frozen. Cells were isolated and cultured from the other half. Protein lysates were made and expression in tissue (T) and cells (C) was compared by western blotting. Because none of the loading controls were stable between cells and tissue we used proteins remaining on the gels for normalization in these experiments. Panel a shows blotting for bladder Thbs4 (top) and proteins on the gel (bottom). Panel b shows summarized data from experiments in ( a ). Panel c shows blotting for Thbs4 in ileum. Panels d through ( f ) show ATF6α-P50 in tissue and cells from bladder, aorta and ileum respectively. Close to 10-fold ATF6α activation was seen in cells compared to tissue in each case. Panels g through ( i ) show the effect of adenoviral overexpression of Thbs4 in mouse bladder SMCs. Western blotting ( g ) showed close to 2000-fold overexpression of Thbs4 ( h ), but no further increase of the ATF6α-P50 level ( i ).

Article Snippet: After protein separation and semi-dry transfer to nitrocellulose for 10–20 min in the Trans-Blot Turbo system, we cut the membranes horizontally to allow for blotting of multiple targets using the following antibodies: thrombospondin-4 (Thbs4, sc-7657-R and sc-390734, Santa Cruz Biotechnology, AF2390, R&D systems and PA5-34386, Thermo Scientific), ATF6α (Atf6, ab11909 (a.k.a.

Techniques: Isolation, Cell Culture, Expressing, Western Blot, Activation Assay, Over Expression

Journal: Scientific Reports

Article Title: Assessing the contribution of thrombospondin-4 induction and ATF6α activation to endoplasmic reticulum expansion and phenotypic modulation in bladder outlet obstruction

doi: 10.1038/srep32449

Figure Lengend Snippet: Thbs4 knockout (KO) mice and littermate wild types (WT) were subjected to outlet obstruction, and bladders were harvested at 7 days. Absolute bladder weights in WT and KO mice were not different either before or following obstruction ( a ). Bladder to body weight ratios were also similar between genotypes, irrespective of obstruction ( b ). Panel c shows western blots for Thbs4, ATF6α-P50 and the ER stress markers Pdi, Calr, Manf and Sdf2l1. ATF6α-P50 formation was reduced in the obstructed KO bladder ( c,d ) but induction of Pdi, Calr and Manf was not affected (panel c and summarized data in ( e ) through ( g )). We also examined contractile markers by western blotting ( h ), and myosin heavy chain, calponin and SM22α were similarly repressed in obstructed WT compared to obstructed KO bladders (( i ) through ( k )).

Article Snippet: After protein separation and semi-dry transfer to nitrocellulose for 10–20 min in the Trans-Blot Turbo system, we cut the membranes horizontally to allow for blotting of multiple targets using the following antibodies: thrombospondin-4 (Thbs4, sc-7657-R and sc-390734, Santa Cruz Biotechnology, AF2390, R&D systems and PA5-34386, Thermo Scientific), ATF6α (Atf6, ab11909 (a.k.a.

Techniques: Knock-Out, Western Blot

Journal: medRxiv

Article Title: Cadherin-26 drives macrophage alternative activation via suppressing STUB1-mediated IL-4Rα ubiquitination in asthma

doi: 10.1101/2024.08.01.24311333

Figure Lengend Snippet: A) Representative images for co-immunofluorescence staining of Cdh26 (green) and Il-4Rα (red) in BALF from mouse (scale bar = 50 μm). Nuclei was stained with DAPI (blue). B) The numbers of Cdh26 + / Il-4Rα + cells in BALF were performed using ImageJ. C-H) The mRNA levels of Arg1 , Cd206 , Fizz1 , Ym1 , Ccl22 and Ccl17 in mouse lungs measured by quantitative PCR. The transcript levels were expressed as log2 transformed and relative to the mean value for control group. I) Representative images for co-immunofluorescence staining of Cd68 (green) and Cd206 (red) in BALF from mouse (scale bar = 50 μm). n=8-10 mice per group. Data are mean ± SD. * p <0.05; ** p <0.01; *** p <0.001. Abbreviations: Cd68, Cd68 molecule; Cd206, mannose receptor C-type 1; Arg1, arginase-1; YM1, also named as Chil3, chitinase-like protein 3; Fizz1, found in inflammatory zone 1; Ccl17, C-C motif chemokine ligand 17; Ccl22, C-C motif chemokine ligand 22; Il-4Rα, lnterleukin-4 receptor subunit alpha.

Article Snippet: Antibodies used in western blotting were: anti-CDH26 mAb (Sigma-Aldrich, 1:1000 dilution), mouse anti-Il-4Rα mAb (R&D system, 1:1000 dilution), human anti-IL-4Rα mAb (Abcam, 1:1000 dilution), anti-IL-2Rγ mAb (Abclonal, 1:1000 dilution), anti-total and phosphorylated Stat6 pAb (Cell Signaling, 1:1,000 dilution), anti-STUB1 mAb (Abcam, 1:1000 dilution), mouse anti-Cd206 mAb (R&D system, 1:1000 dilution) and anti-ARG1 mAb (Proteintech, 1:1000 dilution), anti-FLAG mAb (Proteintech, 1:2000 dilution), anti-GFP mAb (Proteintech, 1:2000 dilution), anti-ubiquitin pAb (Proteintech, 1:1000 dilution), anti-anti-GAPDH pAb (Proteintech, 1:4000 dilution) and anti-anti-ACTB pAb (Proteintech, 1:4000 dilution).

Techniques: Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Transformation Assay, Control

Journal: medRxiv

Article Title: Cadherin-26 drives macrophage alternative activation via suppressing STUB1-mediated IL-4Rα ubiquitination in asthma

doi: 10.1101/2024.08.01.24311333

Figure Lengend Snippet: A) Representative images of immunostaining of CDH26 (red), IL-4Rα (green) and CD206 (yellow) in BAL cells from asthma patients (n=4) and control subjects (n=4). Nuclei was stained with DAPI (blue). B) The numbers of CDH26 + /IL-4Rα + /CD206 + cells were counted in the cytospin of BAL cells in 5 fields for each subject and the average number were calculated using ImageJ (n = 4 per group). C) IL4RA transcript levels in induced sputum cells from asthma patients (n=32) and control subjects (n=14) were determined by quantitative PCR. The transcript levels were expressed as log2 transformed and relative to the mean value for control subjects. D) The expression levels of IL4RA in a microarray data (GSE76262) using sputum cells from subjects with mild-moderate (n=25) and severe asthma (n=49) were analyzed. E-F) The protein levels of Il-4Rα in BMDM cells exposed to Il-4 for 48h were determined by western blotting. Densitometry assay was performed using ImageJ, and Il-4Rα protein levels were indexed to Gapdh. G) The transcript levels of Il4ra in Il-4 stimulated BMDMs derived from WT or Cdh26 −/− mice were determined by quantitative PCR. The transcript levels were expressed as log2 transformed and relative to the mean value for control group. H-I) The protein levels of p-STAT6 in WT or Cdh26 −/− mice derived-BMDM cells stimulated with or without Il-4 for 6h were determined by western blotting. Densitometry assay was performed using ImageJ, and p-STAT6 protein levels were indexed to total STAT6. J) Representative images for immunofluorescence staining of Il-4Rα (green) and Cd206 (red) in BAL cells from Cdh26 fl/fl Lyz2Cre and Cdh26 fl/fl mice challenged with saline or OVA. Nuclei was stained with DAPI (blue). K-L) The numbers of Il-4Rα + Cd206 + cells (K), Il-4Rα + (L) were counted in the cytospin of BAL cells in 5 fields for each mouse and the average number were calculated for Cdh26 fl/fl Lyz2Cre and Cdh26 fl/fl mice challenged with saline or OVA using ImageJ (n = 4 mice per group). M) Representative images for immunofluorescence staining of Il-4Rα (green) and Cd206 (red) in saline- or Il-4-stimulated BMDMs derived from Cdh26 fl/fl Lyz2Cre and Cdh26 fl/fl mice. Nuclei was stained with DAPI (blue). N-O) The numbers of Il-4Rα + Cd206 + (N), Il-4Rα + (O) cells were counted in saline- or Il-4-stimulated BMDMs derived from Cdh26 fl/fl Lyz2Cre and Cdh26 fl/fl mice using ImageJ (n=4 wells per group). Data are mean ± SD. * p <0.05; ** p <0.01; *** p <0.001. Abbreviations: CDH26, cadherin-26; CD206, mannose receptor C-Type 1; IL-4Rα, interleukin-4 receptor subunit alpha; BAL, bronchoalveolar lavage; BMDM, bone marrow derived macrophage; STAT6, signal transducer and activator of transcription 6; Gapdh, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Antibodies used in western blotting were: anti-CDH26 mAb (Sigma-Aldrich, 1:1000 dilution), mouse anti-Il-4Rα mAb (R&D system, 1:1000 dilution), human anti-IL-4Rα mAb (Abcam, 1:1000 dilution), anti-IL-2Rγ mAb (Abclonal, 1:1000 dilution), anti-total and phosphorylated Stat6 pAb (Cell Signaling, 1:1,000 dilution), anti-STUB1 mAb (Abcam, 1:1000 dilution), mouse anti-Cd206 mAb (R&D system, 1:1000 dilution) and anti-ARG1 mAb (Proteintech, 1:1000 dilution), anti-FLAG mAb (Proteintech, 1:2000 dilution), anti-GFP mAb (Proteintech, 1:2000 dilution), anti-ubiquitin pAb (Proteintech, 1:1000 dilution), anti-anti-GAPDH pAb (Proteintech, 1:4000 dilution) and anti-anti-ACTB pAb (Proteintech, 1:4000 dilution).

Techniques: Immunostaining, Control, Staining, Real-time Polymerase Chain Reaction, Transformation Assay, Expressing, Microarray, Western Blot, Derivative Assay, Immunofluorescence, Saline

Journal: medRxiv

Article Title: Cadherin-26 drives macrophage alternative activation via suppressing STUB1-mediated IL-4Rα ubiquitination in asthma

doi: 10.1101/2024.08.01.24311333

Figure Lengend Snippet: A-C) IL-4Rα and IL-2Rγ protein levels were measured in A549 cells transfected with control or CDH26 siRNA and treated with CHX for 0, 6, 12, 24h using western blotting. Densitometry assay was performed using ImageJ, and IL-4Rα and IL-2Rγ protein levels were indexed to GAPDH. D-F) IL-4Rα and IL-2Rγ protein levels were measured in A549 cells transfected with empty or CDH26 overexpressing vector and treated with CHX for 0, 6, 12, 24h using western blotting. Densitometry assay was performed using ImageJ, and IL-4Rα and IL-2Rγ protein levels were indexed to GAPDH. The experiment was repeated 3 times independently. G-H) IL-4Rα protein level was determined in A549 cells transfected with control or CDH26 siRNA and treated with MG132 using western blotting. Densitometry assay was performed using ImageJ, and IL-4Rα protein levels were indexed to GAPDH. I-J) IL-4Rα protein level was determined in A549 cells transfected with control or CDH26 siRNA and treated with CQ using western blotting. Densitometry assay was performed using ImageJ, and IL-4Rα protein level was indexed to GAPDH. K) HEK-293T cells were transfected with plasmids expressing IL-4Rα GFP and CDH26 FLAG as indicated, and cell lysates were immunoprecipitated with anti-GFP antibody followed by western blotting. The ubiquitination level of IL-4Rα was assessed using ubiquitin antibodies. All experiments were repeated for at least three times independently. Data are mean ± SD. * p <0.05. Abbreviations: CDH26, cadherin-26; IL-4Rα, interleukin-4 receptor subunit alpha; IL-2Rγ, interleukin-2 receptor subunit gamma; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CHX, cycloheximide; MG132, Z-Leu-Leu-Leu-al; CQ, chloroquine.

Article Snippet: Antibodies used in western blotting were: anti-CDH26 mAb (Sigma-Aldrich, 1:1000 dilution), mouse anti-Il-4Rα mAb (R&D system, 1:1000 dilution), human anti-IL-4Rα mAb (Abcam, 1:1000 dilution), anti-IL-2Rγ mAb (Abclonal, 1:1000 dilution), anti-total and phosphorylated Stat6 pAb (Cell Signaling, 1:1,000 dilution), anti-STUB1 mAb (Abcam, 1:1000 dilution), mouse anti-Cd206 mAb (R&D system, 1:1000 dilution) and anti-ARG1 mAb (Proteintech, 1:1000 dilution), anti-FLAG mAb (Proteintech, 1:2000 dilution), anti-GFP mAb (Proteintech, 1:2000 dilution), anti-ubiquitin pAb (Proteintech, 1:1000 dilution), anti-anti-GAPDH pAb (Proteintech, 1:4000 dilution) and anti-anti-ACTB pAb (Proteintech, 1:4000 dilution).

Techniques: Transfection, Control, Western Blot, Plasmid Preparation, Expressing, Immunoprecipitation

Journal: medRxiv

Article Title: Cadherin-26 drives macrophage alternative activation via suppressing STUB1-mediated IL-4Rα ubiquitination in asthma

doi: 10.1101/2024.08.01.24311333

Figure Lengend Snippet: A) HEK-293T cells were transfected with plasmids expressing CDH26 FLAG , and cell lysates were immunoprecipitated with anti-FLAG antibody followed by mass spectrum. B) Volcanic map of the proteins interacting with CDH26 detected by mass spectrum. C) Veen diagram shown the protein molecules identified by intersecting the proteins interacting with CDH26 and the proteins involved in ubiquitin-mediated proteolysis. D) STUB1 transcript levels in bronchial brushings from asthma patients (n=24) and control subjects (n=10) were determined using quantitative PCR. E) STUB1 transcript levels in BAL cells from asthma patients (n=32) and control subjects (n=17) were determined using quantitative PCR. F) The transcript levels of Stub1 in lungs of Cdh26 fl/fl Lyz2Cre or Cdh26 fl/fl mice challenged with saline or OVA were determined using quantitative PCR (n = 8-10 mice per group). The transcript levels were expressed as log2 transformed and relative to the mean value of control group. G-H) The protein levels of Stub1 in lungs of Cdh26 fl/fl Lyz2Cre or Cdh26 fl/fl mice challenged with saline or OVA were determined using western blotting (n = 3 per group). Densitometry assay was performed using ImageJ, and Stub1 protein levels were indexed to Actb. I) Binding of CDH26 with STUB1 was examined using immunoprecipitation assay. J) Binding of IL-4Rα with STUB1 was examined using immunoprecipitation assay. K-L) A549 cells were treated with CDH26 siRNA and/or STUB1 siRNA, and IL-4Rα protein levels were determined using western blotting. Densitometry assay was performed using ImageJ, and IL-4Rα protein level was indexed to GAPDH. The experiment was repeated 3 times independently. M) HEK-293T cells were transfected with plasmids expressing IL-4Rα GFP and CDH26 FLAG as indicated, and cell lysates were immunoprecipitated with anti-GFP or IgG antibody followed by western blotting. The ubiquitination level of IL-4Rα was assessed using ubiquitin antibodies. N) The binding interface between CDH26 (716-724, 787-794, and 802-817) and STUB1 was predicted based on the molecular docking model using zdock. O) HEK-293T cells were transfected with plasmids expressing full-CDH26 (1–832) and del-CDH26 FLAG (deleting amino acids 716-724, 787-794, and 802-817), and cell lysates were immunoprecipitated with anti-FLAG or IgG antibody followed by western blotting. P) HEK-293T cells were transfected with plasmids expressing IL-4Rα GFP , full-CDH26 and del-CDH26 FLAG , and cell lysates were immunoprecipitated with anti-GFP or IgG antibody followed by western blotting. Data are mean ± SD. * p <0.05. Abbreviations: CDH26, cadherin-26; STUB1, STIP1 homology and U-box containing protein 1; ACTB, actin beta; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL-4Rα, interleukin-4 receptor subunit alpha.

Article Snippet: Antibodies used in western blotting were: anti-CDH26 mAb (Sigma-Aldrich, 1:1000 dilution), mouse anti-Il-4Rα mAb (R&D system, 1:1000 dilution), human anti-IL-4Rα mAb (Abcam, 1:1000 dilution), anti-IL-2Rγ mAb (Abclonal, 1:1000 dilution), anti-total and phosphorylated Stat6 pAb (Cell Signaling, 1:1,000 dilution), anti-STUB1 mAb (Abcam, 1:1000 dilution), mouse anti-Cd206 mAb (R&D system, 1:1000 dilution) and anti-ARG1 mAb (Proteintech, 1:1000 dilution), anti-FLAG mAb (Proteintech, 1:2000 dilution), anti-GFP mAb (Proteintech, 1:2000 dilution), anti-ubiquitin pAb (Proteintech, 1:1000 dilution), anti-anti-GAPDH pAb (Proteintech, 1:4000 dilution) and anti-anti-ACTB pAb (Proteintech, 1:4000 dilution).

Techniques: Transfection, Expressing, Immunoprecipitation, Control, Real-time Polymerase Chain Reaction, Saline, Transformation Assay, Western Blot, Binding Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Lipopolysaccharide Alters the m6A Epitranscriptomic Tagging of RNAs in Cardiac Tissue

doi: 10.3389/fmolb.2021.670160

Figure Lengend Snippet: M 6 A single-base site qPCR was used to confirm the microarray data for the top four methylated lncRNAs and top six methylated mRNAs in left ventricle tissue between the LPS and control groups. * p < 0.05 versus the Ctrl group. Ctrl, control; LPS, lipopolysaccharide.

Article Snippet: The cRNAs were combined and hybridized onto an Arraystar Rat mRNA&lncRNA Epitranscriptomic Microarray (4 × 44K, Arraystar) that contained 27,770 mRNA and 10,582 lncRNA degenerate probes.

Techniques: Microarray, Methylation, Control

Journal: PLoS ONE

Article Title: Analysis of the Transcriptome in Hyperoxic Lung Injury and Sex-Specific Alterations in Gene Expression

doi: 10.1371/journal.pone.0101581

Figure Lengend Snippet: List of genes selected for validation by qPCR analysis.

Article Snippet: Pdk4 , Mm01166879_m1.

Techniques: Biomarker Discovery

Journal: PLoS ONE

Article Title: Analysis of the Transcriptome in Hyperoxic Lung Injury and Sex-Specific Alterations in Gene Expression

doi: 10.1371/journal.pone.0101581

Figure Lengend Snippet: List of pulmonary DEGs after hyperoxia exposure.

Article Snippet: Pdk4 , Mm01166879_m1.

Techniques:

Journal: PLoS ONE

Article Title: Analysis of the Transcriptome in Hyperoxic Lung Injury and Sex-Specific Alterations in Gene Expression

doi: 10.1371/journal.pone.0101581

Figure Lengend Snippet: Fold change for the selected genes in the microarray and qPCR experiment.

Article Snippet: Pdk4 , Mm01166879_m1.

Techniques: Microarray, Derivative Assay