human prap1  (Proteintech)

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Proline-Rich Acidic Protein 1 (PRAP1) Protects the Gastrointestinal Epithelium From Irradiation-Induced Apoptosis

doi: 10.1016/j.jcmgh.2020.06.011

Figure Lengend Snippet: PRAP1 is an intrinsically disordered protein conserved in placental mammals. ( A ) Amino acid sequence of human PRAP1 with signal peptide and secreted portion of the protein labeled. ( B ) Analysis of the PRAP1 amino acid sequence using the Predictor of Natural Disordered Regions (PONDR) software. The predicted ordered and disordered regions are plotted for each residue. ( C ) Size exclusion chromatogram of purified recombinant PRAP1 protein compared with a molecular weight standard: (a) thyroglobulin (670,000 daltons), (b) γ-globulin (158,000 daltons), (c) ovalbumin (44,000 daltons), (d) myoglobin (17,000 daltons), and (e) vitamin B12 (1350 daltons). ( D ) Circular dichroism spectra of recombinant PRAP1. ( E ) Analysis of the human PRAP1 amino acid sequence using the comparative genomics feature of Ensembl software. The number of PRAP1 orthologs identified in each taxonomic clade are indicated.

Article Snippet: Cell lysates were blotted with a commercially available antibody specific for human PRAP1 (Proteintech, first blot) or with PRAP1 rabbit antisera generated using 6xHis-PRAP1 (second blot). ( C ) Western blot of small intestine and uterine whole tissue from wild-type and Prap1 -/- mice, blotted with PRAP1 antisera introduced in panel B . ( D ) Immunofluorescence staining of wild-type and Prap1 -/- duodenum using PRAP1 antisera.

Techniques: Sequencing, Labeling, Software, Residue, Purification, Recombinant, Molecular Weight, Circular Dichroism

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Proline-Rich Acidic Protein 1 (PRAP1) Protects the Gastrointestinal Epithelium From Irradiation-Induced Apoptosis

doi: 10.1016/j.jcmgh.2020.06.011

Figure Lengend Snippet: Generation and validation of PRAP1 recombinant protein, PRAP1 antisera, and Prap1 -/- mice. ( A ) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis with Coomassie staining of recombinant 6xHis-PRAP1 expressed in E coli and purified by a Ni-NTA affinity chromatography column followed by a size exclusion column. ( B ) A human colonic epithelial cell line (SK-CO15) was transfected to overexpress human (H) and mouse (M) PRAP1. Cell lysates were blotted with a commercially available antibody specific for human PRAP1 (Proteintech, first blot) or with PRAP1 rabbit antisera generated using 6xHis-PRAP1 (second blot). ( C ) Western blot of small intestine and uterine whole tissue from wild-type and Prap1 -/- mice, blotted with PRAP1 antisera introduced in panel B . ( D ) Immunofluorescence staining of wild-type and Prap1 -/- duodenum using PRAP1 antisera. Whole-body knockout mice were procured from MMRRC-UC Davis and backcrossed to obtain a fully congenic C57BL/6 background. Scale bar : 100 μm. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KO, knockout, SI, small intestine; WT, wild-type; UT, uterine tissue.

Article Snippet: Cell lysates were blotted with a commercially available antibody specific for human PRAP1 (Proteintech, first blot) or with PRAP1 rabbit antisera generated using 6xHis-PRAP1 (second blot). ( C ) Western blot of small intestine and uterine whole tissue from wild-type and Prap1 -/- mice, blotted with PRAP1 antisera introduced in panel B . ( D ) Immunofluorescence staining of wild-type and Prap1 -/- duodenum using PRAP1 antisera.

Techniques: Biomarker Discovery, Recombinant, Polyacrylamide Gel Electrophoresis, Staining, Purification, Affinity Column, Transfection, Generated, Western Blot, Immunofluorescence, Knock-Out

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Proline-Rich Acidic Protein 1 (PRAP1) Protects the Gastrointestinal Epithelium From Irradiation-Induced Apoptosis

doi: 10.1016/j.jcmgh.2020.06.011

Figure Lengend Snippet: PRAP1 is highly expressed by the epithelium of the gastrointestinal tract in mice and human beings. ( A ) Quantification of Prap1 transcript measured via quantitative PCR in the indicated tissues from 8-week-old wild-type C57BL/6 mice (n = 3 mice). ( B ) Western blot analysis for the detection of PRAP1 protein abundance in the indicated tissues dissected from 8-week-old wild-type C57BL/6 mice. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Images are representative of 3 mice per tissue collected. ( C ) Immunofluorescence for the detection of PRAP1 (green) in the duodenum of 8-week-old wild-type C57BL/6 mice or Prap1 -/- mice. Images are representative of the analysis of 5 mice per tissue collected. ( D ) Immunofluorescence staining of PRAP1 (green) in the duodenum of 8-week-old wild-type mice at 60× magnification. ( E and F ) Immunohistochemistry staining for the detection of PRAP1 (brown) in the human ileum ( E ) and colon ( F ). Image is representative of 3 subjects. Prox, proximal.

Article Snippet: Cell lysates were blotted with a commercially available antibody specific for human PRAP1 (Proteintech, first blot) or with PRAP1 rabbit antisera generated using 6xHis-PRAP1 (second blot). ( C ) Western blot of small intestine and uterine whole tissue from wild-type and Prap1 -/- mice, blotted with PRAP1 antisera introduced in panel B . ( D ) Immunofluorescence staining of wild-type and Prap1 -/- duodenum using PRAP1 antisera.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Quantitative Proteomics, Control, Immunofluorescence, Staining, Immunohistochemistry

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Proline-Rich Acidic Protein 1 (PRAP1) Protects the Gastrointestinal Epithelium From Irradiation-Induced Apoptosis

doi: 10.1016/j.jcmgh.2020.06.011

Figure Lengend Snippet: Prap1 -/- mice have an altered microbiota in the small intestine. ( A ) The body weight of wild-type and Prap1 -/- littermates at 10 weeks old. ( B ) H&E staining of wild-type and Prap1 -/- small intestine. Images are representative of 3 mice per group. ( C ) Quantification of villi and crypt length in the small intestine of wild-type and Prap1 -/- mice. Significance was determined using an unpaired t test (n = 3 mice). ∗ P < .05. ( D and E ) Quantitative PCR analysis of Pcna ( D ) and Bax ( E ) expression in whole tissue from the small intestine of wild-type and Prap1 -/- mice. Expression levels are relative to Gapdh . Significance was determined using an unpaired t test. ∗ P < .05 (n = 6 mice). ( F ) Pie chart comparison of the average Bacteriodetes:Firmicutes phyla ratio in the small intestine of wild-type and Prap1 -/- littermates measured via 16S ribosomal RNA sequencing (n ≥ 12 mice). ( G ) Relative abundance of Firmicutes and Bacteroidetes in the small intestine of wild-type and Prap1 -/- littermates. Significance was determined using an unpaired t test (n ≥ 12 mice). ∗ P < .05. All data are graphed as the means ± SEM. Bax RA, Bax relative abundance; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KO, knockout; Pcna RA, Pcna relative abundance; WT, wild-type.

Article Snippet: Cell lysates were blotted with a commercially available antibody specific for human PRAP1 (Proteintech, first blot) or with PRAP1 rabbit antisera generated using 6xHis-PRAP1 (second blot). ( C ) Western blot of small intestine and uterine whole tissue from wild-type and Prap1 -/- mice, blotted with PRAP1 antisera introduced in panel B . ( D ) Immunofluorescence staining of wild-type and Prap1 -/- duodenum using PRAP1 antisera.

Techniques: Staining, Real-time Polymerase Chain Reaction, Expressing, Comparison, RNA Sequencing, Knock-Out

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Proline-Rich Acidic Protein 1 (PRAP1) Protects the Gastrointestinal Epithelium From Irradiation-Induced Apoptosis

doi: 10.1016/j.jcmgh.2020.06.011

Figure Lengend Snippet: Prap1 -/- mice have increased inflammation but no significant intestinal barrier defect. ( A ) A multiplex enzyme-linked immunosorbent assay (ELISA) was used for the detection of 10 proinflammatory cytokines in sera of 10-week-old wild-type and Prap1 -/- littermates. Data are shown as a heat map, with red indicating a higher than average concentration. Each column represents 1 mouse. ( B ) Graphic representation of significantly different cytokine levels shown in panel A , including IL2, IL4, and IL12p70. Statistical significance was determined using an unpaired t test (n = 5 mice). ∗ P < .05. ( C ) Quantification of IL12β transcript levels measured via quantitative PCR in the colon of 10-week-old wild-type and Prap1 -/- mice relative to the abundance of β-actin. Statistical significance was determined using an unpaired t test (n = 5 mice). ∗ P < .05. ( D ) Quantification of IgA levels in fecal pellets collected from 10-week-old wild-type and Prap1 -/- mice, measured via ELISA. Statistical significance was determined using an unpaired t test (n = 5 mice). ( E ) Quantification of FITC dextran in the sera of unchallenged 10-week-old wild-type and Prap1 -/- mice 4 hours after oral gavage with 4 kilodaltons FITC dextran. Statistical significance was determined using an unpaired t test (n = 5 mice). IFN, interferon; KC, keratinocyte chemoattractant; RA, relative abundance; TNF, tumor necrosis factor.

Article Snippet: Cell lysates were blotted with a commercially available antibody specific for human PRAP1 (Proteintech, first blot) or with PRAP1 rabbit antisera generated using 6xHis-PRAP1 (second blot). ( C ) Western blot of small intestine and uterine whole tissue from wild-type and Prap1 -/- mice, blotted with PRAP1 antisera introduced in panel B . ( D ) Immunofluorescence staining of wild-type and Prap1 -/- duodenum using PRAP1 antisera.

Techniques: Multiplex Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Real-time Polymerase Chain Reaction

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Proline-Rich Acidic Protein 1 (PRAP1) Protects the Gastrointestinal Epithelium From Irradiation-Induced Apoptosis

doi: 10.1016/j.jcmgh.2020.06.011

Figure Lengend Snippet: Prap1 -/- mice are more susceptible to radiologic challenge and have increased apoptosis in the intestinal epithelium. ( A ) Percentage body weight loss of wild-type C57BL/6 and littermate Prap1 -/- mice after 10 Gy TBI. Statistical analysis represents a comparison of wild-type vs Prap1 -/- on each respective day using 2-way analysis of variance, Bonferroni multiple comparisons test (n = 4 mice). ∗ P < .05, ∗∗ P < .01. ( B ) Survival of wild-type, Prap1 +/- or Prap1 -/- mice after 10 Gy TBI. Statistical significance was determined using the log-rank test (n ≥ 11 mice). ∗∗ P < .01. ( C ) Representative images of terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-positive cells (green) within the small intestine of 8-week-old wild-type and Prap1 -/- littermates 6 hours after receiving 10 Gy TBI. ( D ) Representative images of cleaved caspase-3–positive cells (green) within the small intestine of 8-week-old wild-type and Prap1 -/- littermates 72 hours after receiving 10 Gy TBI. ( E ) Quantification of TUNEL-positive cells in panel C . Significance was determined using an unpaired t test (n ≥ 11 mice). ∗∗ P < .01. ( F ) Quantification of cleaved caspase-3–positive cells in panel F . Significance was determined using an unpaired t test (n ≥ 6 mice). ∗∗ P < .01. ( G ) Quantification of Bax transcript via quantitative PCR on whole tissue from the small intestine of wild-type and Prap1 -/- littermates 96 hours after 10 Gy TBI. Significance was determined using an unpaired t test (n = 6 mice). ∗ P < .05. ( H ) Quantification of Pcna transcript via quantitative PCR on whole tissue from the small intestine of wild-type and Prap1 -/- littermates 96 hours after 10 Gy TBI. Significance was determined using an unpaired t test (n = 6 mice). ( I ) Quantification of serum FITC dextran in wild-type and Prap1 -/- littermates after oral gavage with 4 kilodaltons FITC dextran 72 hours after 10 Gy TBI. Significance was determined using an unpaired t test (n ≥ 3 mice). All data are graphed as the means ± SEM. ( J ) Quantification of Prap1 transcript via quantitative PCR on whole tissue from the small intestine of wild-type mice at different time points after 10 Gy TBI. Significance was determined using 1-way analysis of variance, Tukey multiple comparisons test (n = 6 mice). ∗∗ P < .005. Bax RA, Bax relative abundance; DAPI, 4′,6-diamidino-2-phenylindole; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; KO, knockout; Pcna RA, Pcna relative abundance; WT, wild-type.

Article Snippet: Cell lysates were blotted with a commercially available antibody specific for human PRAP1 (Proteintech, first blot) or with PRAP1 rabbit antisera generated using 6xHis-PRAP1 (second blot). ( C ) Western blot of small intestine and uterine whole tissue from wild-type and Prap1 -/- mice, blotted with PRAP1 antisera introduced in panel B . ( D ) Immunofluorescence staining of wild-type and Prap1 -/- duodenum using PRAP1 antisera.

Techniques: Comparison, End Labeling, TUNEL Assay, Real-time Polymerase Chain Reaction, Knock-Out

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Proline-Rich Acidic Protein 1 (PRAP1) Protects the Gastrointestinal Epithelium From Irradiation-Induced Apoptosis

doi: 10.1016/j.jcmgh.2020.06.011

Figure Lengend Snippet: PRAP1 protects enteroids from irradiation-induced apoptosis by limiting p21 expression. ( A ) The percentage of viability of wild-type or Prap1 -/- enteroids 48 hours after 2 Gy. Cell viability was measured using the addition of MTT and the percentage of viability was calculated using the cell viability measured before irradiation. Significance was determined using an unpaired t test (n ≥ 7 wells). ∗ P < .05. ( B ) Representative immunofluorescence images for the detection of cleaved caspase-3 in wild-type and Prap1 -/- enteroids 24 hours after 2 Gy. Examples of cleaved caspase-3–positive enteroids are indicated by a white arrowhead . Scale bar : 1000 μm. ( C ) Quantification of cleaved caspase-3–positive enteroids in panel B . Each data point represents the percentage of cleaved caspase-3–positive enteroids in a well. Data were pooled from 4 independent experiments. Significance was determined via unpaired t test (n = 4 mice per group). ∗∗ P < .01. ( D–F ) Quantification of Prap1 ( D ), p21 ( E ), and p18 ( F ) transcript via quantitative PCR in wild-type and Prap1 -/- enteroids 24 hours after 0 Gy and 1 Gy. Significance was determined via an unpaired t test. ∗ P < .05. Each data point represents enteroids harvested from a unique mouse (n = 3 mice per group). ( G ) Protein levels were determined via Western blot from epithelial cells transfected with an empty cytomegalovirus expression vector (pCMV) pCMV or a cytomegalovirus expression vector encoding PRAP1 (pCMV-PRAP1) 48 hours after 8 Gy. ( H and I ) Quantification of p21 ( H ) and p18 ( I ) protein levels in panel G determined via signal intensity relative to GAPDH. Significance was determined using an unpaired t test (n = 4). ∗ P > .05. All data are graphed as means ± SEM. Casp3, caspase-3; DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KO, knockout; pCMV, empty cytomegalovirus expression vector; WT, wild-type

Article Snippet: Cell lysates were blotted with a commercially available antibody specific for human PRAP1 (Proteintech, first blot) or with PRAP1 rabbit antisera generated using 6xHis-PRAP1 (second blot). ( C ) Western blot of small intestine and uterine whole tissue from wild-type and Prap1 -/- mice, blotted with PRAP1 antisera introduced in panel B . ( D ) Immunofluorescence staining of wild-type and Prap1 -/- duodenum using PRAP1 antisera.

Techniques: Irradiation, Expressing, Immunofluorescence, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Plasmid Preparation, Knock-Out