Journal: Carcinogenesis

Article Title: The lung-enriched p53 mutants V157F and R158L/P regulate a gain of function transcriptome in lung cancer

doi: 10.1093/carcin/bgz087

Figure Lengend Snippet: uPA protein expression is mutp53-dependent. Protein expression of uPA encoded by the RNA-seq identified gene PLAU, was assessed in p53-depleted and control conditions. (A) Western blot of A549, H2087, H441, H2110 and H2009 cells transfected with negative control or siRNA directed against p53 (25 nM). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is shown as a loading control. (B) qRT–PCR was performed to evaluate mRNA expression of PLAU after depletion of p53 by siRNA. mRNA expression values are normalized to GAPDH and siCtrl condition within each cell line. (C) Protein and conditioned media were harvested from cells after overnight incubation in reduced-serum conditions. Nitrocellulose membranes were stained with Ponceau S dye to visualize protein bands for loading control. Protein expression of pro-uPA (49 kDa) and its cleaved and activated forms B-chain (33 kDa) and A-chain (19 kDa) are shown in cell lysates and conditioned media. (D) Protein and E. mRNA expression of uPA (PLAU) following depletion of V157F mutp53 by siRNA.

Article Snippet: The following antibodies were used to detect protein expression by western blot: p53 (DO-1, sc-126; Santa Cruz), p21 (C-19, sc-397, Santa Cruz) and uPA (AF1310, R&D systems).

Techniques: Expressing, RNA Sequencing, Control, Western Blot, Transfection, Negative Control, Quantitative RT-PCR, Incubation, Staining

Journal: Circulation

Article Title: Releasing the Brakes on the Fibrinolytic System in Pulmonary Emboli: Unique Effects of Plasminogen Activation and α2-Antiplasmin Inactivation

doi: 10.1161/CIRCULATIONAHA.116.024421

Figure Lengend Snippet: Lung tissue containing FITC-fibrin labeled (green) pulmonary emboli was immunostained (red) to detect the expression of (A) tPA, (B) uPA and (C) plasminogen (Pg) (D) The total immune-stained area (arbitrary units; a.u) for each protein was measured in a 20× (100 μm) image of an embolized vs non embolized (Control; dashed yellow outline) pulmonary artery in the lungs. n=3, mean ± SEM. **p<0.01; ns, non-significant.

Article Snippet: The primary antibodies include rabbit anti-mouse against tPA (ASMTPA-GF; Molecular Innovations), uPA (urokinase plasminogen activator) (ASMUPA-GF-HT; Molecular Innovations) and PAI-1 (IASMPAI-GF; Innovative Research), rabbit anti-mouse plasminogen (Abcam), goat anti-mouse α2-antiplasmin (AF1239; R&D Systems), and rat anti-mouse Ly6G (clone1A8, #127602; Biolegend).

Techniques: Labeling, Expressing, Staining

Journal: Journal of leukocyte biology

Article Title: Involvement of fibrinolytic regulators in adhesion of monocytes to vascular endothelial cells induced by glycated LDL and to aorta from diabetic mice.

doi: 10.1189/jlb.0513262

Figure Lengend Snippet: Figure 2. Effects of glyLDL on the expression of uPA, uPAR, LRP, P-selectin, ICAM-1, and MCP-1 and the impact of uPA on PAI-1 in EC. (A and B) HUVEC were treated with vehicle [control (ctr)], 50– 150 g/ml glyLDL (gLDL) for 1–12 h for uPA. (C) HUVEC were treated with 1 g/ml uPA or vehi- cle for 30 min, and the incubation was continued by adding vehicle of 100 g/ml glyLDL for 24 h. (D and E) HUVEC were treated with vehicle or 100 g/ml glyLDL for 1–24 h for ICAM-1, P-selectin (P-sel), and MCP-1. Abundances of uPA, uPAR, LRP, ICAM-1, P-selectin, MCP-1, PAI-1, and -actin in EC were analyzed using Western blotting and assessed semiquantitatively using ChemiDoc software. Values were expressed in folds of control after normalization with -actin (meansd; n3 independent experiments). sal., Saline. *P 0.05; **P 0.01 versus vehicle control; P 0.01 versus glyLDL.

Article Snippet: Targeted proteins in total cellular proteins of EC were analyzed using Western blotting analysis, as described previously [5]. mAb or polyclonal antibodies against human PAI-1, uPA, uPAR, HSF1, p22-phox, ICAM-1, P-selectin, monocyte adhesion protein (MCP)-1, LRP, or -actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) or Abcam (Cambridge, UK).

Techniques: Expressing, Control, Incubation, Western Blot, Software, Saline

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