rabbit anti odc1 antibody  (Bioss)

Journal: bioRxiv

Article Title: Polyamines sustain epithelial regeneration in aged intestines by modulating protein homeostasis

doi: 10.1101/2024.07.26.605278

Figure Lengend Snippet: a Schematic representation of dynamics of protein synthesis comparing our damage/regeneration model to the fasting/re-feeding model from Imada et al. 2024. b Schematic representation of the polyamine biosynthesis and ornithine metabolism pathways. Red proteins are indicating the rate limiting enzymes in these pathways. c Protein abundance profiles of the enzymes involved in polyamines biosynthetic pathways. d LC-MS quantification of polyamines from crypts lysate. Each dot represents one mouse. n =4 mice per condition. Error bars represent the SD. P -value was calculated using Welch’s t-test for time point comparison and two-way ANOVA for overall day and age comparisons. e Upper: representative immunoblot for hypusinated EIF5A and total EIF5A proteins. Lower: quantification of hypusinated EIF5A and total EIF5A normalized to ponceau staining. Each dot represents one mouse. n =3-5 mice per each indicated condition. Error bars represent the SD. P -value was calculated using Welch’s t-test for time point comparison and two-way ANOVA for overall day and age comparisons. f Distribution of proteins enriched in hypusinated EF5A-dependent motifs across clusters that show distinct dynamics in young and old mice (see for cluster profiles). Highlighted are proteins that contain the highest number of hypusinated EIF5A-dependent motifs. P -value was calculated using Wilcoxon rank sum test. g Protein abundance profiles of the COL1A1, COL15A1 and FBN2 proteins in tissue lysate from indicated groups. h Upper: Schematic of ODC1 KO induction and 5-FU treatment. Lower: Relative body weight of young ODC1 KO and WT mice treated with a single dose of 5-FU. Body weight of each mouse was normalized to its body weight at the day of injection. n =4 ODC1 KO, n =3 ODC1 WT mice. Error bars represent the SD. P -value was calculated using Welch’s t-test for time point comparison and two-way ANOVA for overall day, treatment and day x treatment comparisons.

Article Snippet: Membranes were then stained with Ponceau S (Sigma #P7170-1L) for 5 min on a shaker (Heidolph Duomax 1030), washed with Milli-Q water, imaged on a Molecular Imager ChemiDocTM XRS + Imaging system (Bio-Rad Laboratories CA, USA) and destained by 2 washes with PBS and 2 washes in TBST (Tris-buffered saline (TBS, 25 mM Tris, 75 mM NaCl), with 0.5% (v/v) Tween-20) for 5 min. After incubation for 5 min in EveryBlot blocking buffer (Bio-Rad Laboratories #12010020), membranes were incubated overnight with primary antibodies: Mouse-Puromycin (Millipore Sigma #MABE343), Rabbit-Ubiquitin linkage specific K48 (Abcam #ab140601), Rabbit-SQSTM1/p62 (Abcam #ab91526), Mouse-S6 ribosomal protein (Cell Signaling Technologies #2317), Mouse-Ubiquitin P4D1 (Santa Cruz Biotechnology #sc-8017), Rabbit-Cleaved Caspase 3 (Cell Signaling Technologies #9661), Rabbit-Caspase 3 (Cell Signaling Technologies #9662), Rabbit-phospho H3 (Cell Signaling Technologies # 9701), Rabbit-Hypusinated EIF5A (Millipore Sigma #ABS1064), Mouse-EIF5a (BD Biosciences #611977), Rabbit-ODC1 (Abcam #ab137679) diluted (1:1000) in enzyme dilution buffer (0.2% (w/v) BSA, 0.1% (v/v) Tween20 in PBS) at 4 °C on a tube roller (BioCote® Stuart® SRT6).

Techniques: Liquid Chromatography with Mass Spectroscopy, Comparison, Western Blot, Staining, Injection

Journal: bioRxiv

Article Title: Polyamines sustain epithelial regeneration in aged intestines by modulating protein homeostasis

doi: 10.1101/2024.07.26.605278

Figure Lengend Snippet: a Protein abundance profiles of the enzymes involved in urea cycle metabolism. b Quantification of amino acids by LC-MS. Each dot represents one mouse. n =4 mice per condition. Error bars represent the SD. P -value was calculated using Welch’s t-test for time point comparison and two-way ANOVA for overall day and age comparisons. c Protein abundance profiles of the Eukaryotic Translation Initiation Factor 5A (EIF5A) d Top 29 motifs that depend on hypusinated EIF5A for efficient translation according to . e Immunoblot for ODC1. Proteins were extracted from formalin-fixed paraffin embedded (FFPE) whole intestinal tissue samples. Bands corresponding to the dimeric (catalytically active form ) and monomeric forms of ODC1 are highlighted. f Upper: Schematic of ODC1 KO induction and PBS treatment. Lower: Relative body weight of young ODC1 KO and WT mice treated with a single dose of 5-FU. Body weight of each mouse was normalized to its body weight at the day of injection. n =3 ODC1 KO, n =2 ODC1 WT mice. Black line indicates the mean with SD for each day in each treatment group. P -value is calculated by two-way ANOVA for overall day, treatment and day x treatment comparisons.

Article Snippet: Membranes were then stained with Ponceau S (Sigma #P7170-1L) for 5 min on a shaker (Heidolph Duomax 1030), washed with Milli-Q water, imaged on a Molecular Imager ChemiDocTM XRS + Imaging system (Bio-Rad Laboratories CA, USA) and destained by 2 washes with PBS and 2 washes in TBST (Tris-buffered saline (TBS, 25 mM Tris, 75 mM NaCl), with 0.5% (v/v) Tween-20) for 5 min. After incubation for 5 min in EveryBlot blocking buffer (Bio-Rad Laboratories #12010020), membranes were incubated overnight with primary antibodies: Mouse-Puromycin (Millipore Sigma #MABE343), Rabbit-Ubiquitin linkage specific K48 (Abcam #ab140601), Rabbit-SQSTM1/p62 (Abcam #ab91526), Mouse-S6 ribosomal protein (Cell Signaling Technologies #2317), Mouse-Ubiquitin P4D1 (Santa Cruz Biotechnology #sc-8017), Rabbit-Cleaved Caspase 3 (Cell Signaling Technologies #9661), Rabbit-Caspase 3 (Cell Signaling Technologies #9662), Rabbit-phospho H3 (Cell Signaling Technologies # 9701), Rabbit-Hypusinated EIF5A (Millipore Sigma #ABS1064), Mouse-EIF5a (BD Biosciences #611977), Rabbit-ODC1 (Abcam #ab137679) diluted (1:1000) in enzyme dilution buffer (0.2% (w/v) BSA, 0.1% (v/v) Tween20 in PBS) at 4 °C on a tube roller (BioCote® Stuart® SRT6).

Techniques: Liquid Chromatography with Mass Spectroscopy, Comparison, Western Blot, Formalin-fixed Paraffin-Embedded, Injection

Journal: BMC Research Notes

Article Title: Identification of unique expression signatures and therapeutic targets in esophageal squamous cell carcinoma

doi: 10.1186/1756-0500-5-73

Figure Lengend Snippet: Protein expression assessments of POSTN, ODC1, and ASPA . Immunohistochemical staining of POSTN ( A ), ODC1 ( B ), and ASPA ( C ) are shown, with rabbit IgG ( D ) as the negative control. The left and middle panels for each represent tumor area at the magnification of 40× and 10×. The right panel for each represents the tumor adjacent normal area at a magnification of 10×

Article Snippet: Antibodies used included rabbit polyclonal anti-ODC1 primary antibody (Sigma) at 1:200, rabbit polyclonal anti-POSTN primary antibody (Sigma) at 1:300, rabbit polyclonal anti-ASPA primary antibody (Abcam) 1:300, and prediluted rabbit polyclonal anti-IMP3 primary antibody (Abcam).

Techniques: Expressing, Immunohistochemical staining, Staining, Negative Control

Journal: BioMed Research International

Article Title: Immunohistochemical Expression of Ornithine Decarboxylase, Diamine Oxidase, Putrescine, and Spermine in Normal Canine Enterocolic Mucosa, in Chronic Colitis, and in Colorectal Cancer

doi: 10.1155/2015/172756

Figure Lengend Snippet: Expression of DAO and ODC in different colonic pathological conditions. (a) Morphological aspect of colonic carcinoma with areas of squamous metaplasia. (b) The same section stained with anti-DAO polyclonal antibody; note the weak positivity diffused particularly in well-preserved mucosal epithelium. (c) In a consequent section stained with anti-ODC pAb the strong expression of the enzyme is observed in metaplastic undifferentiated areas. (d) Morphology of granulomatous colitis with severe crypts involvement and areas of subepithelial fibrosis. (e) The expression of DAO in a consequent section shows a continuous and strong expression in the superficial epithelium, but only occasional and spotted strong stain in epithelium lining the crypts. (f) Weak and focal expression of ODC in a successive section evidences the low concentration of the enzyme in colonic epithelium during the granulomatous phlogosis. ((a) and (d) H&E; (b), (c), (e), and (f) IHC stain, Meyer's haematoxylin counterstain; (a), (b), and (c) bar = 300 µ m; (d), (e), and (f) bar = 600 µ m).

Article Snippet: Tissue sections were incubated overnight in a moist chamber at 4°C with different primary antibodies (Abs): rabbit polyclonal Ab against ODC ( Bioss antibodies , pAb rb-anti ODC antibody, bs-1294R ; diluted 1 : 50), DAO ( Biorbyt ; pAb rb-anti DAO antibody, orb192676 ; diluted 1 : 100), PUT ( Thermo Fisher Scientific ; pAb rb-anti Pentane-1,5-diamine, #PA1-86537, diluted 1 : 20), and SPM ( Abcam , ab7318, diluted 1 : 20) antigens.

Techniques: Expressing, Staining, Concentration Assay

Journal: Cancers

Article Title: A G316A Polymorphism in the Ornithine Decarboxylase Gene Promoter Modulates MYCN-Driven Childhood Neuroblastoma

doi: 10.3390/cancers13081807

Figure Lengend Snippet: Analysis of AG clones generated from the GG SK-N-BE(2)-C cell line by CRISPR-Cas9 editing. ( A ) The CRISPR-Cas9 editing strategy was adopted to edit SK-N-BE(2)-C cells. The gRNA cleavage site (inverted triangles) is located 9 bp upstream of the G316A SNP. The donor DNA sequence carries the A SNP mutation (black). ( B , C ) ODC1 expression analysis by qRT-PCR ( B ) and Western blot ( C ) of CRISPR-edited clones AG-1 and AG-2, compared to parental SK-N-BE(2)-C cells. qRT-PCR data were normalized using GUSB as reference gene, and standardized as previously described . Β-actin was used as the loading control for the Western blot. Uncropped WB images are available in . ( D ) Proliferation rates of CRISPR-edited clones. Cell growth was measured by BrdU assay. Absorbance data were normalized to day 1 and SK-N-BE(2)-C was used as the control. ( E ) Analysis of the effect of DFMO on CRISPR-edited clones by clonogenic assay. The total area occupied by cell colonies was measured by crystal violet staining and subsequent ImageJ analysis. All p values were determined by one-way ANOVA tests and compared to SK-N-BE(2)-C as control. ( F ) ChIP analysis of H3-histone acetylation of the G316A SNP spanning region in the CRISPR-edited clones. The analyzed sequences in the ODC1 locus are E-box1, G316A SNP, Exon 9 and a sequence located +1500 bp downstream of the locus. Acetylation of a region in the Actin gene was used as a control. Enrichment data were normalized to a non-acetylated region located 15,000 bp upstream of the ODC1 locus. A representation of the analyzed region is shown in . ** p < 0.005, *** p < 0.001. All experiments were performed at least 3 times.

Article Snippet: Rabbit monoclonal anti-Odc1 (Abcam, Cambridge, UK; ab126590) and rabbit monoclonal anti-β-Actin (Sigma-Aldrich, St. Louis, MO, USA; a2066) antibodies were used.

Techniques: Clone Assay, Generated, CRISPR, Sequencing, Mutagenesis, Expressing, Quantitative RT-PCR, Western Blot, BrdU Staining, Clonogenic Assay, Staining

Journal: Cancers

Article Title: A G316A Polymorphism in the Ornithine Decarboxylase Gene Promoter Modulates MYCN-Driven Childhood Neuroblastoma

doi: 10.3390/cancers13081807

Figure Lengend Snippet: The effect of the ODC1 SNP on MYCN binding and ODC1 promoter activity. ( A,B ) MYCN binding was assessed using EMSA with SK-N-BE(2)-C ( A ) and Tet21N ( B ) nuclear extracts. Nuclear extracts were incubated in the presence of radiolabeled probes containing either the G SNP (G probe) or the A SNP (A probe), and E-box 3. Competition analysis was performed by incubating the binding reaction with 5–10-fold and 10–25-fold molar excess of unlabeled G probe carrying either a wild type (WT) or Mutant E-box. Quantification of the specific bands (arrow) is shown in . Note that the order of the lanes differs between A and B. ( C ) Luciferase reporter assays in Tet21N cells transfected with a reporter plasmid containing a region of the ODC1 promoter that includes either the G or the A SNP. Untreated Tet21N cells with MYCN overexpression (MYCN+) were compared to tetracycline-treated cells (MYCN−), where MYCN overexpression is blocked. Replicates were standardized as previously described . A two-way ANOVA was used to test for an interaction between the effects of the SNP and MYCN expression on promoter activity (SNP: F 1,16 = 20.0, p < 0.001; MYCN expression: F 1,16 = 203.4, p < 0.001; Interaction: F 1,16 = 13.0, p = 0.002) followed by Tukey’s post-hoc test for multiple pairwise comparisons. A representation of all the constructs utilized for these experiments are shown in . *** p < 0.001.

Article Snippet: Rabbit monoclonal anti-Odc1 (Abcam, Cambridge, UK; ab126590) and rabbit monoclonal anti-β-Actin (Sigma-Aldrich, St. Louis, MO, USA; a2066) antibodies were used.

Techniques: Binding Assay, Activity Assay, Incubation, Mutagenesis, Luciferase, Transfection, Plasmid Preparation, Over Expression, Expressing, Construct

Journal: Cancers

Article Title: A G316A Polymorphism in the Ornithine Decarboxylase Gene Promoter Modulates MYCN-Driven Childhood Neuroblastoma

doi: 10.3390/cancers13081807

Figure Lengend Snippet: G316A ODC1 promoter SNP and its prognostic significance in colorectal and lung cancer. ( A ) In an online colorectal cancer dataset of 226 primary samples (Sieber dataset), low expression of ODC1 was prognostic of poor outcome (dichotomized at the median). This dataset is available on the R2 platform (R2: Genomics Analysis and Visualization Platform, https://hgserver1.amc.nl/cgi-bin/r2/main.cgi , accessed on 3 August 2020). Patients at risk are not shown as they cannot be generated using this platform. ( B ) In a cohort of 63 rectal cancer patients, where most patients had a favorable outcome, the AA/AG genotype tended to have a poorer overall outcome than the GG genotype. NS—not significant. ( C ) In a lung cancer cohort of 524 squamous cell carcinoma patients, low ODC1 expression is associated with worse outcome. Data was obtained from KM Plotter . ( D ) In a second cohort of 161 squamous cell carcinomas patients, those with AG/AA genotypes tended to have a worse outcome compared with GG genotypes.

Article Snippet: Rabbit monoclonal anti-Odc1 (Abcam, Cambridge, UK; ab126590) and rabbit monoclonal anti-β-Actin (Sigma-Aldrich, St. Louis, MO, USA; a2066) antibodies were used.

Techniques: Expressing, Generated