antibody against hb egf  (Bioss)

Journal: The journal of sexual medicine

Article Title: Heparin-binding epidermal growth factor-like growth factor improves erectile function in streptozotocin-induced diabetic mice.

doi: 10.1093/jsxmed/qdae079

Figure Lengend Snippet: Figure 1. Expression of HB-EGF is decreased under diabetic conditions. (A) Double-immunofluorescence staining of PECAM-1/HB-EGF (top panel) and NG2/HB-EGF (bottom panel) in penile tissues of age-matched control and diabetic mice. Nuclei were stained with DAPI (blue). Scale bar, 100 μm. (B) Representative Western blots for PECAM-1, NG2, and HB-EGF in corpus cavernosum tissue from age-matched control and diabetic mice. (C) Representative Western blots for HB-EGF in MCECs, and MCPs exposed to NG and HG conditions. (D-F) Normalized areas of PECAM-1, NG2, and HB-EGF–immunopositive staining were quantified using ImageJ software (n = 4). (G-K) Normalized band intensity values for indicated targets were quantified using ImageJ software (n = 4). Results are presented as mean ± SEM. ∗∗P < .01; ∗∗∗P < .001. The relative ratio of the control or NG group was arbitrarily set to 1. DAPI, 4′,6-diamidino-2-phenylindole; DM, diabetes mellitus; HB-EGF, heparin-binding epidermal growth factor–like growth factor; HG, high glucose; MCEC, mouse cavernous endothelial cell; MCP, mouse cavernous pericyte; NG, normal glucose; PECAM-1, platelet/endothelial adhesion molecule 1.

Article Snippet: For immunofluorescence analysis, penis tissues were fixed in 4% paraformaldehyde at 4◦C for 24 hours, and cell samples were fixed in 4% paraformaldehyde at 4◦C for 15 minutes at room temperature, as described previously.21 Frozen tissue sections (12 μm thick), MCECs, and MCPs were washed and then incubated overnight at 4◦C with the following antibodies: primary antibody against HB-EGF (1:100, Santa Cruz Biotechnology Inc., Dallas, TX, United States), platelet/endothelial adhesion molecule 1 (PECAM1; 1:100; Millipore, Temecula, CA, United States), NG2 (1:50; Millipore), phosphohistone H3 (1:50; Millipore), neuronal NOS (nNOS; 1:100; Santa Cruz Biotechnology Inc.), neurofilament (1:50; Sigma-Aldrich), occludin (1:100; Novus Biologicals, Littleton, CO, United States), claudin-5 (1:100; Invitrogen, Carlsbad, CA, United States), oxidized low-density lipoprotein (Ox-LDL: 1:100; Abcam, Cambridge, MA, United States), and phospho-eNOS (1:50; Invitrogen).

Techniques: Expressing, Staining, Control, Western Blot, Software, Binding Assay

Journal: The journal of sexual medicine

Article Title: Heparin-binding epidermal growth factor-like growth factor improves erectile function in streptozotocin-induced diabetic mice.

doi: 10.1093/jsxmed/qdae079

Figure Lengend Snippet: Figure 2. HB-EGF promotes angiogenesis and proliferation under HG conditions. (A) Tube formation assays of MCECs treated with PBS or HB-EGF (50, 100, 200, or 500 ng/mL) under normal-glucose (NG) and high-glucose (HG) conditions. Scale bar = 100 μm. (B and C) Migration assays in MCECs and MCPs treated with PBS or HB-EGF (100 ng/mL) under NG or HG conditions. Scale bar = 100 μm. (D) Representative images of immunofluorescent staining of PH3 (red and green) in MCECs and MCPs treated with PBS or HB-EGF (100 ng/mL) under NG or HG conditions. Nuclei were stained with DAPI (blue). Scale bar, 50 μm. (F) The number of master junctions in MCECs was quantified using ImageJ software (n = 4). (G and H) The number of migrated MCECs (G) and MCPs (H) within the frame line was quantified using ImageJ software (n = 4). (I and J) The number of PH3-positive MCECs (I) and MCPs (J) was quantified using ImageJ software (n = 4). Results are presented as mean ± SEM. ∗∗P < .01; ∗∗∗P < .001. The relative ratio of the NG (G-J) group was arbitrarily set to 1. DAPI, 4′,6-diamidino-2-phenylindole; HB-EGF, heparin-binding epidermal growth factor–like growth factor; HG, high glucose; MCEC, mouse cavernous endothelial cell; MCP, mouse cavernous pericyte; NG, normal glucose; PBS, phosphate-buffered saline; PH3, phosphohistone H3.

Article Snippet: For immunofluorescence analysis, penis tissues were fixed in 4% paraformaldehyde at 4◦C for 24 hours, and cell samples were fixed in 4% paraformaldehyde at 4◦C for 15 minutes at room temperature, as described previously.21 Frozen tissue sections (12 μm thick), MCECs, and MCPs were washed and then incubated overnight at 4◦C with the following antibodies: primary antibody against HB-EGF (1:100, Santa Cruz Biotechnology Inc., Dallas, TX, United States), platelet/endothelial adhesion molecule 1 (PECAM1; 1:100; Millipore, Temecula, CA, United States), NG2 (1:50; Millipore), phosphohistone H3 (1:50; Millipore), neuronal NOS (nNOS; 1:100; Santa Cruz Biotechnology Inc.), neurofilament (1:50; Sigma-Aldrich), occludin (1:100; Novus Biologicals, Littleton, CO, United States), claudin-5 (1:100; Invitrogen, Carlsbad, CA, United States), oxidized low-density lipoprotein (Ox-LDL: 1:100; Abcam, Cambridge, MA, United States), and phospho-eNOS (1:50; Invitrogen).

Techniques: Migration, Staining, Software, Binding Assay, Saline

Journal: The journal of sexual medicine

Article Title: Heparin-binding epidermal growth factor-like growth factor improves erectile function in streptozotocin-induced diabetic mice.

doi: 10.1093/jsxmed/qdae079

Figure Lengend Snippet: Figure 3. HB-EGF improves erectile function in STZ-induced diabetic mice. (A) Representative ICP responses in age-matched control and diabetic mice 2 weeks after 2 intracavernous administrations of PBS or various doses of HB-EGF (days −3 and 0; 1 μg, 5 μg, and 10 μg in 20 μL of PBS, respectively). The solid bar indicates the stimulus interval. (B and C) ratios of maximum ICP (B) and total ICP (C) to MSBP were calculated for each group (n = 10). Results are presented as mean ± SEM. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. HB-EGF, heparin-binding epidermal growth factor–like growth factor; ICP, intracavernous pressure; MSBP, mean systolic blood pressure; PBS, phosphate-buffered saline; STZ, streptozotocin.

Article Snippet: For immunofluorescence analysis, penis tissues were fixed in 4% paraformaldehyde at 4◦C for 24 hours, and cell samples were fixed in 4% paraformaldehyde at 4◦C for 15 minutes at room temperature, as described previously.21 Frozen tissue sections (12 μm thick), MCECs, and MCPs were washed and then incubated overnight at 4◦C with the following antibodies: primary antibody against HB-EGF (1:100, Santa Cruz Biotechnology Inc., Dallas, TX, United States), platelet/endothelial adhesion molecule 1 (PECAM1; 1:100; Millipore, Temecula, CA, United States), NG2 (1:50; Millipore), phosphohistone H3 (1:50; Millipore), neuronal NOS (nNOS; 1:100; Santa Cruz Biotechnology Inc.), neurofilament (1:50; Sigma-Aldrich), occludin (1:100; Novus Biologicals, Littleton, CO, United States), claudin-5 (1:100; Invitrogen, Carlsbad, CA, United States), oxidized low-density lipoprotein (Ox-LDL: 1:100; Abcam, Cambridge, MA, United States), and phospho-eNOS (1:50; Invitrogen).

Techniques: Control, Binding Assay, Saline

Journal: The journal of sexual medicine

Article Title: Heparin-binding epidermal growth factor-like growth factor improves erectile function in streptozotocin-induced diabetic mice.

doi: 10.1093/jsxmed/qdae079

Figure Lengend Snippet: Figure 4. HB-EGF increases cavernous endothelial cell, pericyte, and neuronal cell content in STZ-induced diabetic mice. (A and B) double-immunofluorescence staining of PECAM-1/NG2 (A) and nNOS/NF (B) in penile tissues from age-matched control and diabetic mice 2 weeks after two intracavernous administrations of PBS or HB-EGF (days −3 and 0; 5 μg in 20 μL of PBS). Nuclei were stained with DAPI (blue). Scale bar, 100 μm (A) or 25 μm (B). (C-F) quantification of PECAM-1, NG2, nNOS, and NF immunopositive areas using ImageJ software (n = 4). (G) Representative Western blots for PECAM-1, NG2, and nNOS in penile tissues from age-matched control and diabetic mice 2 weeks after two intracavernous administrations of PBS or HB-EGF (days −3 and 0; 5 μg in 20 μL of PBS). (H-J) normalized band intensity values for indicated targets were quantified using ImageJ software (n = 4). Results are presented as mean ± SEM. ∗∗P < .01; ∗∗∗P < .001. The relative ratio of the control group was arbitrarily set to 1. DAPI, 4′,6-diamidino-2-phenylindole; PECAM-1, platelet/endothelial adhesion molecule 1; HB-EGF, heparin-binding epidermal growth factor–like growth factor; NG2, nerve/glial-antigen 2; STZ, streptozotocin.

Article Snippet: For immunofluorescence analysis, penis tissues were fixed in 4% paraformaldehyde at 4◦C for 24 hours, and cell samples were fixed in 4% paraformaldehyde at 4◦C for 15 minutes at room temperature, as described previously.21 Frozen tissue sections (12 μm thick), MCECs, and MCPs were washed and then incubated overnight at 4◦C with the following antibodies: primary antibody against HB-EGF (1:100, Santa Cruz Biotechnology Inc., Dallas, TX, United States), platelet/endothelial adhesion molecule 1 (PECAM1; 1:100; Millipore, Temecula, CA, United States), NG2 (1:50; Millipore), phosphohistone H3 (1:50; Millipore), neuronal NOS (nNOS; 1:100; Santa Cruz Biotechnology Inc.), neurofilament (1:50; Sigma-Aldrich), occludin (1:100; Novus Biologicals, Littleton, CO, United States), claudin-5 (1:100; Invitrogen, Carlsbad, CA, United States), oxidized low-density lipoprotein (Ox-LDL: 1:100; Abcam, Cambridge, MA, United States), and phospho-eNOS (1:50; Invitrogen).

Techniques: Staining, Control, Software, Western Blot, Binding Assay

Journal: The journal of sexual medicine

Article Title: Heparin-binding epidermal growth factor-like growth factor improves erectile function in streptozotocin-induced diabetic mice.

doi: 10.1093/jsxmed/qdae079

Figure Lengend Snippet: Figure 5. HB-EGF induces tight junction protein levels and reduces permeability in STZ-induced diabetic mice. (A) Double-immunofluorescence staining of occludin (green) and claudin-5 (red) in penile tissues from age-matched control and diabetic mice 2 weeks after two intracavernous administrations of PBS or HB-EGF (days −3 and 0; 5 μg in 20 μL of PBS). (B) Ox-LDL (green) staining in the same samples. Nuclei labeled with DAPI (blue). Scale bar, 100 μm. (C-E) quantitative analysis of occludin, claudin, and ox-LDL immunopositive areas using ImageJ software (n = 4). (F) Representative Western blots for Occludin and Claudin-5 in penile tissues from age-matched control and diabetic mice 2 weeks after two intracavernous administrations of PBS or HB-EGF (days −3 and 0; 5 μg in 20 μL of PBS). (G and H) normalized band intensity values for indicated targets were quantified using ImageJ software (n = 4). Results are presented as mean ± SEM. ∗∗P < .01; ∗∗∗P < .001. The relative ratio of the control group was arbitrarily set to 1. DAPI, 4′,6-diamidino-2-phenylindole; DM, diabetes mellitus; HB-EGF, heparin-binding epidermal growth factor–like growth factor; ox-LDL, oxidized low-density lipoprotein; STZ, streptozotocin.

Article Snippet: For immunofluorescence analysis, penis tissues were fixed in 4% paraformaldehyde at 4◦C for 24 hours, and cell samples were fixed in 4% paraformaldehyde at 4◦C for 15 minutes at room temperature, as described previously.21 Frozen tissue sections (12 μm thick), MCECs, and MCPs were washed and then incubated overnight at 4◦C with the following antibodies: primary antibody against HB-EGF (1:100, Santa Cruz Biotechnology Inc., Dallas, TX, United States), platelet/endothelial adhesion molecule 1 (PECAM1; 1:100; Millipore, Temecula, CA, United States), NG2 (1:50; Millipore), phosphohistone H3 (1:50; Millipore), neuronal NOS (nNOS; 1:100; Santa Cruz Biotechnology Inc.), neurofilament (1:50; Sigma-Aldrich), occludin (1:100; Novus Biologicals, Littleton, CO, United States), claudin-5 (1:100; Invitrogen, Carlsbad, CA, United States), oxidized low-density lipoprotein (Ox-LDL: 1:100; Abcam, Cambridge, MA, United States), and phospho-eNOS (1:50; Invitrogen).

Techniques: Permeability, Staining, Control, Labeling, Software, Western Blot, Binding Assay

Journal: The journal of sexual medicine

Article Title: Heparin-binding epidermal growth factor-like growth factor improves erectile function in streptozotocin-induced diabetic mice.

doi: 10.1093/jsxmed/qdae079

Figure Lengend Snippet: Figure 6. HB-EGF activates the AKT/eNOS signaling pathway in STZ-induced diabetic mice. (A) Representative Western blots for p-AKTSer473, total AKT, p-eNOSSer1177, and total eNOS in penile tissues from age-matched control and diabetic mice 2 weeks after two intracavernous administrations of PBS or HB-EGF (days −3 and 0; 5 μg in 20 μL of PBS). (B and C) normalized band intensity values for indicated targets were quantified using ImageJ software (n = 4). (D) Double-immunofluorescence staining of occludin (green) and claudin-5 (red) in penile tissues after ICP study. (E) Quantitative analysis of p-eNOSSer1177 immunopositive areas using ImageJ software (n = 4). Results are presented as mean ± SEM. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. The relative ratio of the control group was arbitrarily set to 1. DAPI, 4′,6-diamidino-2-phenylindole; eNos, endothelial nitric oxide synthase; HB-EGF, heparin-binding epidermal growth factor–like growth factor; ICP, intracavernous pressure; PBS, phosphate-buffered saline; p-eNOS, phosphorylated endothelial nitric oxide synthase; STZ, streptozotocin.

Article Snippet: For immunofluorescence analysis, penis tissues were fixed in 4% paraformaldehyde at 4◦C for 24 hours, and cell samples were fixed in 4% paraformaldehyde at 4◦C for 15 minutes at room temperature, as described previously.21 Frozen tissue sections (12 μm thick), MCECs, and MCPs were washed and then incubated overnight at 4◦C with the following antibodies: primary antibody against HB-EGF (1:100, Santa Cruz Biotechnology Inc., Dallas, TX, United States), platelet/endothelial adhesion molecule 1 (PECAM1; 1:100; Millipore, Temecula, CA, United States), NG2 (1:50; Millipore), phosphohistone H3 (1:50; Millipore), neuronal NOS (nNOS; 1:100; Santa Cruz Biotechnology Inc.), neurofilament (1:50; Sigma-Aldrich), occludin (1:100; Novus Biologicals, Littleton, CO, United States), claudin-5 (1:100; Invitrogen, Carlsbad, CA, United States), oxidized low-density lipoprotein (Ox-LDL: 1:100; Abcam, Cambridge, MA, United States), and phospho-eNOS (1:50; Invitrogen).

Techniques: Western Blot, Control, Software, Staining, Binding Assay, Saline

Journal: Reproductive biology and endocrinology : RB&E

Article Title: microRNA-194 is increased in polycystic ovary syndrome granulosa cell and induce KGN cells apoptosis by direct targeting heparin-binding EGF-like growth factor.

doi: 10.1186/s12958-021-00850-w

Figure Lengend Snippet: Fig. 3 HB-EGF was directly targeted by miR-194. A Venn diagram demonstrates the intersection of data extracted from several bioinformatics websites. (B) Binding sites shared between miR-194 and HB-EGF. (C-D) HB-EGF protein and mRNA expression in the GCs. KGN cells were transfected for 48 h with miR-194 mimic, miR-194 inhibitor, or the miR-NC group. (E) Luciferase reporter assays. (F) miR-194 and HB-EGF mRNA expression in PCOS patient ovarian GCs. n= 3-6 for each group; *P < 0.05 vs. control group or miR-NC group

Article Snippet: Non-fat milk was used to treat membranes for 2 h at 4 °C before probing overnight with antibodies against HB-EGF (catalog # 15,071; Cell Signaling Technology, USA), p53 (catalog # wL01919; Wanleigio), p21 (catalog # WLH0362; Wanleigio), p16 (catalog # WLH3673; Wanleigio) and β-actin (catalog # 3700; Cell Signaling Technology).

Techniques: Binding Assay, Expressing, Transfection, Luciferase, Control

Journal: Reproductive biology and endocrinology : RB&E

Article Title: microRNA-194 is increased in polycystic ovary syndrome granulosa cell and induce KGN cells apoptosis by direct targeting heparin-binding EGF-like growth factor.

doi: 10.1186/s12958-021-00850-w

Figure Lengend Snippet: Fig. 4 HB-EGF was decreased in ovarian tissues from PCOS rat model and PCOS GCs. (A) Western blotting and (B) qRT-PCR were used to quantify HB-EGF protein and mRNA levels in the PCOS rat model. (C) qRT-PCR demonstrate HB-EGF mRNA expression in PCOS patient GCs and the control group (n=10). B-C. 𝑛 = 3-6 for each group. *P < 0.05 vs. control group

Article Snippet: Non-fat milk was used to treat membranes for 2 h at 4 °C before probing overnight with antibodies against HB-EGF (catalog # 15,071; Cell Signaling Technology, USA), p53 (catalog # wL01919; Wanleigio), p21 (catalog # WLH0362; Wanleigio), p16 (catalog # WLH3673; Wanleigio) and β-actin (catalog # 3700; Cell Signaling Technology).

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Control

Journal: Reproductive biology and endocrinology : RB&E

Article Title: microRNA-194 is increased in polycystic ovary syndrome granulosa cell and induce KGN cells apoptosis by direct targeting heparin-binding EGF-like growth factor.

doi: 10.1186/s12958-021-00850-w

Figure Lengend Snippet: Fig. 5 Upregulated HB-EGF reverses the effect of miR-194 mimic in KGN cells growth and apoptosis. (A) The expression of HB-EGF in KGN cells post-transfection with HB-EGF-OE. (B) The expression of HB-EGF in KGN cells post transfection with miR-194 mimic or miR-194 mimic+HB-EGF-OE. (C-D) The apoptotic rate of KGN cells was determined using AO/EB and flow cytometry. 𝑛 = 3-6 for each group; *P < 0.05 vs. miR-NC mimic group

Article Snippet: Non-fat milk was used to treat membranes for 2 h at 4 °C before probing overnight with antibodies against HB-EGF (catalog # 15,071; Cell Signaling Technology, USA), p53 (catalog # wL01919; Wanleigio), p21 (catalog # WLH0362; Wanleigio), p16 (catalog # WLH3673; Wanleigio) and β-actin (catalog # 3700; Cell Signaling Technology).

Techniques: Expressing, Transfection, Flow Cytometry