nb100-310  (Bio-Techne corporation)

Journal: Cardiovascular Research

Article Title: Deficiency of myeloid PHD proteins aggravates atherogenesis via macrophage apoptosis and paracrine fibrotic signalling

doi: 10.1093/cvr/cvab152

Figure Lengend Snippet: PHDs correlated with in human plaque inflammation. ( A ) Representative pictures of haematoxylin/eosin (HE), PHD1, PHD2, and CD68 in adjacent human carotid plaque sections from different stages [intimal thickening (IT), pathological intimal thickening (PIT), thick fibrous cap atheroma (TkFCA), and intraplaque haemorrhage (IPH)], ( B ) Representative pictures of HE, Scramble, PHD3 in situ hybridization, and CD68 immunoreactivity in IPH human carotid plaque. ( C ) PHD mRNA expression in microarrays of non-diseased arteries (nd, n = 10) and carotid plaques (cp, n = 127) from the BiKE cohort. Carotid plaques were further stratified as asymptomatic (as, n = 40) or symptomatic patients (s, n = 87). ( D ) PHD isoforms mRNA expression in bone marrow-derived macrophages (BMDMs) measured by quantitative PCR ( n = 3–4 replicates). Expression relative to 18S. Statistical analyses were performed using two-way ANOVA, with Bonferroni post hoc test ( C and D ). All results show mean ± SEM. * P < 0.05, *** P < 0.001.

Article Snippet: Slides were incubated with antibodies against PHD1 (Novus Biologicals NB100-310), PHD2 (Novus Biologicals NB100-2219) and CD68 (DAKO, M0814).

Techniques: In Situ Hybridization, Expressing, Derivative Assay, Real-time Polymerase Chain Reaction

Journal: Nature Communications

Article Title: Prolyl hydroxylase substrate adenylosuccinate lyase is an oncogenic driver in triple negative breast cancer

doi: 10.1038/s41467-019-13168-4

Figure Lengend Snippet: Substrate-trapping strategy identifies ADSL as an EglN2 substrate in TNBC. a Immunoblots (IB) of whole-cell extracts (WCE) and immunoprecipitations (IP) of lysates from T47D cells infected with lentivirus encoding either wild-type (WT) or catalytically dead mutant (H358A) FLAG- and HA-tagged EglN2, and treated as indicated overnight. b IB of WCE and IP of lysates from MDA-MB-231 cells infected with lentivirus encoding either EglN2-WT or -H358A, and treated as indicated overnight. c Schematic representation of the hydroxylase (here EglN2) substrate screen. d IB of input (1% of the protein amount used for the pull-down) and GST pull-down of lysates from MDA-MB-231 cells treated as indicated overnight. e IB of WCE and IP of lysates from MDA-MB-231 cells transfected with indicated plasmids and treated as indicated overnight. f Endogenous EglN2 and ADSL IP in MDA-MB-231 treated with DMOG. g , h , i IB of input and GST pull-down of in vitro-translated ADSL ( g , i ) and recombinant EglN2 ( h ). In b , c , d , and e , hypoxia was 1% O 2 and DMOG concentration was 1 mM. Source data are provided as a Source Data file

Article Snippet: Rabbit antibody against EglN2 (NB100-310, dilution 1:2000) was from Novus Biologicals.

Techniques: Western Blot, Infection, Mutagenesis, Transfection, In Vitro, Recombinant, Concentration Assay

Journal: Nature Communications

Article Title: Prolyl hydroxylase substrate adenylosuccinate lyase is an oncogenic driver in triple negative breast cancer

doi: 10.1038/s41467-019-13168-4

Figure Lengend Snippet: ADSL is hydroxylated by EglN2 on Proline 24. a In vitro hydroxylation of GST-ADSL in the presence or absence of recombinant EglN2. Bar graph represents the normalized ratio of the intensity of the oxidized P24-containing peptide to that of ADSL full protein. Error bars represent SEM, n = 4, * P < 0.05 was calculated using two-tailed Student’s t -test. b Fragmentation spectrum of proline hydroxylated peptide YASPEMCFVFSDR detected following experimental procedure described in ( a ). c , d IB of WCE and IP of lysates from 293T cells transfected with the indicated plasmids, and treated as indicated overnight. e IB of lysates from MDA-MB-231 cells overexpressing the indicated dox-inducible ADSL, transduced and treated as indicated. f Representative image of 2-D proliferation of MDA-MB-231 cells overexpressing the indicated dox-inducible ADSL, transduced and treated as indicated. g Enzymatic activity of WT, P24A or R426H ADSL in the presence of its substrate adenylosuccinate (SAMP) or succinylaminoimidazole carboxamide ribotide (SAICAR). Bar graphs represent the normalized percentage of ADSL activity compared with WT ADSL. Error bars represent SEM, n = 4, * P < 0.05, ** P < 0.01, *** P < 0.001 were calculated using one-way ANOVA followed by Tukey’s multiple comparison test. Source data are provided as a Source Data file

Article Snippet: Rabbit antibody against EglN2 (NB100-310, dilution 1:2000) was from Novus Biologicals.

Techniques: In Vitro, Recombinant, Two Tailed Test, Transfection, Activity Assay, Comparison

Journal: Nature Communications

Article Title: Prolyl hydroxylase substrate adenylosuccinate lyase is an oncogenic driver in triple negative breast cancer

doi: 10.1038/s41467-019-13168-4

Figure Lengend Snippet: ADSL controls cMYC negative regulator MIR22HG expression. a Scatterplot of differential-expression results: −log10 FDR-adjusted P -values from comparisons of each gRNA versus control using DESeq2 are plotted for all genes regardless of significance. b , c MIR22HG mRNA expression upon ADSL depletion in ( b ) MDA-MB-231 and ( c ) MDA-MB-436 cells. Graphs represent the mean ± SEM, n = 3 ( b ) and n = 4 ( c ). d Pearson correlation between the expression of ADSL and MIR22HG in TNBC patients from TCGA dataset. e MIR22HG mRNA expression and f cMYC protein level after transfecting MDA-MB-231 cells with either MIR22HG expressing plasmid or empty vector. g MIR22HG mRNA expression and h cMYC protein level after transfecting MDA-MB-231 cells with either three independent siRNAs targeting MIR22HG or siRNA control. i MIR22HG mRNA expression, j 2-D, and k 3-D colony formation in the presence or absence of doxycycline (dox) in MDA-MB-231 cells transduced with dox-inducible MIR22HG expressing lentivirus. Graphs represent the mean ± SEM from four independent sets of samples ( i ), and from two independent experiments, each performed in duplicate ( k ). l , m MIR22HG mRNA expression in ADSL control or knockout MDA-MB-231 cells with or without the expression of ( l ) WT and ( m ) P24A ADSL. n MIR22HG mRNA expression in ADSL control or knockout MDA-MB-231 cells treated as indicated (adenosine concentration was 50 µM). Graphs in ( l ), ( m ), and ( n ) represent the mean ± SEM, n = 3. o Schematic of the proposed mechanism by which EglN2-hydroxylated ADSL controls cMYC and cMYC target gene expression. * P < 0.05, ** P < 0.01, *** P < 0.001 were calculated using one-way ANOVA followed by Dunnett’s multiple comparison test in ( b ), ( c ), and ( g ). In ( e ), ( i ), and ( k ), P -values were calculated using two-tailed Student’s t -test. In ( l ), ( m ), and ( n ), P -values were calculated using one-way ANOVA followed by Tukey’s multiple comparison test. Source data are provided as a Source Data file

Article Snippet: Rabbit antibody against EglN2 (NB100-310, dilution 1:2000) was from Novus Biologicals.

Techniques: Expressing, Quantitative Proteomics, Control, Plasmid Preparation, Transduction, Knock-Out, Concentration Assay, Targeted Gene Expression, Comparison, Two Tailed Test