pro ngf  (Cusabio)

Journal: Frontiers in Pharmacology

Article Title: Jia-Wei-Kai-Xin-San, an Herbal Medicine Formula, Ameliorates Cognitive Deficits via Modulating Metabolism of Beta Amyloid Protein and Neurotrophic Factors in Hippocampus of Aβ 1-42 Induced Cognitive Deficit Mice

doi: 10.3389/fphar.2019.00258

Figure Lengend Snippet: Jia-Wei-Kai-Xin-San treatment induced transformation of neurotrophic factors precursors into mature forms in hippocampus of Aβ injection mice. (A) The treatment of JWKXS and huperzine A in the mice were same as that in . The hippocampus tissues were collected after 1-week treatment and the expressions of proNGF and NGF were analyzed by western blot analysis and ELISA kits. Afterward, the ratio of mNGF to proNGF was calculated. (B) Expressions of proBDNF and BDNF were determined and the ratio of BDNF to proBDNF was calculated as same as that of NGF. Comparisons between groups were carried out by a one-way ANOVA followed by a post hoc Bonferroni test. Values were expressed in the percentage of control group as Mean ± SEM ( n = 8). # p < 0.05 (compared with sham group); ∗ p < 0.05, ∗∗ p < 0.01 (compared with model group).

Article Snippet: Aβ amounts were determined by amyloid beta protein mouse ELISA Kit (Jinyibai Company, China). proNGF and NGF levels were determined by mouse proNGF ELISA kit and mouse NGF ELISA kit (CUSABIO, Houston, TX, United States). proBDNF and BDNF levels were determined by mouse proBDNF ELISA kit and mouse BDNF ELISA kit (Aviscera Bioscience, Santa Clara, CA, United States).

Techniques: Transformation Assay, Injection, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: The Journal of Clinical Investigation

Article Title: Role of proNGF/p75 signaling in bladder dysfunction after spinal cord injury

doi: 10.1172/JCI97837

Figure Lengend Snippet: (A) ProNGF is released into the urine almost immediately after spinal cord transection in mice. The blots were probed with anti-proNGF and pan-NGF antibodies. There was little mature NGF detectable in the urine at these time points. (B) Quantification of proNGF by Western blotting. Within-subject comparison was significant at P = 0.003 based on repeated measures of 1-way ANOVA after Greenhouse-Geisser correction, which resulted in P < 0.01. Subsequent pairwise comparisons were made using Bonferroni correction. (C) Quantification of proNGF detected in mouse urine by proNGF-specific ELISA. Note that mature NGF was not detected by mature NGF-specific ELISA. (D) Immunoprecipitation/Western analyses of urine samples illustrate that the bands detected with proNGF antibody in A are indeed proNGF. Mouse urine samples were immunoprecipitated with 27/21 mature NGF antibody and probed with proNGF antibody. (E) ProNGF is detected in the urine after contusion injuries in mice. (F) ProNGF is present in the urine after spinal cord transection in rats. Mature NGF was not detected by mature NGF-specific ELISA. (G) ProNGF was also detected in human urine after SCI. The upper blot was probed with proNGF-specific antibody (*artifact), while the lower blot was probed with pan-NGF antibody (H-20). Note that recombinant proNGF and mature NGF were included as controls. Based on human proNGF-specific ELISA, the amount of proNGF was 1 and 46 pg/mg for 3 and 6 hours after injury, respectively. Note that 3- and 6-hour urine samples were collected from 2 different individuals. ELISA assays also failed to detect mature NGF as in Western blotting. C, urine from healthy control without SCI.

Article Snippet: ELISA kits for detecting mouse mature and proNGF as well as human proNGF were purchased from Biosensis.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Recombinant

Journal: The Journal of Clinical Investigation

Article Title: Role of proNGF/p75 signaling in bladder dysfunction after spinal cord injury

doi: 10.1172/JCI97837

Figure Lengend Snippet: (A) ProNGF is released into the urine almost immediately after spinal cord transection in mice. The blots were probed with anti-proNGF and pan-NGF antibodies. There was little mature NGF detectable in the urine at these time points. (B) Quantification of proNGF by Western blotting. Within-subject comparison was significant at P = 0.003 based on repeated measures of 1-way ANOVA after Greenhouse-Geisser correction, which resulted in P < 0.01. Subsequent pairwise comparisons were made using Bonferroni correction. (C) Quantification of proNGF detected in mouse urine by proNGF-specific ELISA. Note that mature NGF was not detected by mature NGF-specific ELISA. (D) Immunoprecipitation/Western analyses of urine samples illustrate that the bands detected with proNGF antibody in A are indeed proNGF. Mouse urine samples were immunoprecipitated with 27/21 mature NGF antibody and probed with proNGF antibody. (E) ProNGF is detected in the urine after contusion injuries in mice. (F) ProNGF is present in the urine after spinal cord transection in rats. Mature NGF was not detected by mature NGF-specific ELISA. (G) ProNGF was also detected in human urine after SCI. The upper blot was probed with proNGF-specific antibody (*artifact), while the lower blot was probed with pan-NGF antibody (H-20). Note that recombinant proNGF and mature NGF were included as controls. Based on human proNGF-specific ELISA, the amount of proNGF was 1 and 46 pg/mg for 3 and 6 hours after injury, respectively. Note that 3- and 6-hour urine samples were collected from 2 different individuals. ELISA assays also failed to detect mature NGF as in Western blotting. C, urine from healthy control without SCI.

Article Snippet: ELISA kits for detecting mouse mature and proNGF as well as human proNGF were purchased from Biosensis.

Techniques: Western Blot, Comparison, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Recombinant, Control

Journal: Frontiers in molecular neuroscience

Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward.

doi: 10.3389/fnmol.2016.00063

Figure Lengend Snippet: FIGURE 1 | IP and WB of transgenic and wild-type mice. (A): IP and WB of cortex extracts from male (M) and female (F) TgProNGF#72 and wild-type (WT) mice. IP on extracts from cortex (CTX) with anti-NGF αD11 antibody, followed by WB with anti-NGF or anti-proNGF antibody, as described in Tiveron et al. (2013). A representative WB probed with anti-proNGF (PAb Alomone), (top) or anti-NGF M20 (Santa Cruz) (bottom) is shown. TgproNGF#72 and wild type mice, male and female, were analyzed. (B) Quantitative analysis of proNGF and mature NGF in the CTX of TgproNGF#72 mice, male and female, by IP and WB and densitometric analysis. After anti-NGF IP, the proNGF bands, (in WB probed with anti-proNGF), and the NGF bands, (in the WB probed with anti-NGF antibody), both identified also by Mass Spectrometry, were quantified. The resulting intensities were normalized against the area of the bands, and then compared with an internal standard of recombinant proNGF and NGF. Loaded samples were in the linear range of detection. Comparison between proNGF and NGF amounts in TgproNGF#72, male and female, is reported in the histogram. The experiment was carried out in triplicate.

Article Snippet: The CUSABIO Mouse Pro-Nerve Growth Factor (proNGF) ELISA Kit was purchased and tested.

Techniques: Transgenic Assay, Mass Spectrometry, Recombinant, Comparison

Journal: Frontiers in molecular neuroscience

Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward.

doi: 10.3389/fnmol.2016.00063

Figure Lengend Snippet: FIGURE 3 | NGF and proNGF spiked in the sample buffer (supplied by each supplier of the commercial kits). The histograms summarize the results of the NGF and proNGF spiked separately and together into the assay buffer of the three different commercial kits for the detection of NGF analyzed. The Promega kit was tested in the two different formats: rat mAb Promega and αD11. Two concentrations of recombinant NGF (10 and 100 pg/ml) and proNGF (50 and 200 pg/ml) were spiked into the assay buffer either alone or together with NGF and measured by using the kits previously described. (A) (Emax Promega with mAb Promega), (B) (Emax Promega with mAb αD11), (C) (Chemikine Chemicon), (D) (NGF Rapid ELISA Biosensis) report the values of concentration, interpolated by the calibration curves. The calculated values of the samples spiked with both NGF and proNGF were compared to the spiked value of only NGF. The t-student test was carried out and the p-values were calculated. Two asterisks on the histograms mean a p < 0.001.

Article Snippet: The CUSABIO Mouse Pro-Nerve Growth Factor (proNGF) ELISA Kit was purchased and tested.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Frontiers in molecular neuroscience

Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward.

doi: 10.3389/fnmol.2016.00063

Figure Lengend Snippet: FIGURE 4 | ELISA for the differential detection of NGF and ProNGF. (A) Strategy: capture NGF and measure proNGF (Exploiting the mAb αD11 fast kinetics). ELISA sandwiches format based on a pre-capturing of NGF by a treatment of the sample with mAb αD11 (Cattaneo et al., 1988) on a solid support. Subsequent (Continued)

Article Snippet: The CUSABIO Mouse Pro-Nerve Growth Factor (proNGF) ELISA Kit was purchased and tested.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Frontiers in molecular neuroscience

Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward.

doi: 10.3389/fnmol.2016.00063

Figure Lengend Snippet: FIGURE 5 | SPR analysis—calibration curves obtained with proNGF and NGF over the panel of different antibodies. (A) Different proNGF concentrations tested on anti-proNGF mAb FPro10. Concentrations in nM (from top): 500, 333, 222, 148, 98, 65, 33, 16, 8, 4, 2, 1, 0.5; (B) Different proNGF concentrations tested on anti-proNGF mAb Millipore. Concentrations in nM (from top): 500, 333, 222, 148, 98, 65, 33, 16, 8, 4, 2, 1, 0.5; (C) Different proNGF concentrations tested on anti-NGF mAb αD11. Concentrations in nM (from top): 500, 333, 222, 148, 98, 65, 33, 16, 8, 4, 2, 1, 0.5; (D) Different proNGF concentrations tested on anti-NGF mAb R&D. Concentrations in nM (from top): 100, 50, 25, 12.50, 6.25, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1; (E) Different NGF concentrations tested on anti-NGF mAb R&D. Concentrations in nM (from top): 100, 50, 25, 12.50, 6.25, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1.

Article Snippet: The CUSABIO Mouse Pro-Nerve Growth Factor (proNGF) ELISA Kit was purchased and tested.

Techniques:

Journal: Frontiers in molecular neuroscience

Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward.

doi: 10.3389/fnmol.2016.00063

Figure Lengend Snippet: FIGURE 6 | Binding analysis of NGF and proNGF mixtures to different antibodies. The following antibodies were immobilized on the chip for SPR experiments. (A) anti-proNGF mAb FPro10; (B) anti-proNGF mAb Millipore; (C) anti-NGF mAb αD11. In all the panels, the curves represent the following analytes from top to bottom: 200 nM proNGF + 20 nM NGF (solid blue line, experimental value); 200 nM proNGF (solid yellow line, experimental value); 200 nM proNGF + 20 nM NGF (Continued)

Article Snippet: The CUSABIO Mouse Pro-Nerve Growth Factor (proNGF) ELISA Kit was purchased and tested.

Techniques: Binding Assay

Journal: Frontiers in molecular neuroscience

Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward.

doi: 10.3389/fnmol.2016.00063

Figure Lengend Snippet: FIGURE 7 | AlphaLISA experiments: curves of NGF, proNGF, mix of NGF + proNGF, comparison at different incubation times. Curves of NGF, proNGF, mix of NGF + proNGF, linearly interpolated. NGF and proNGF were assayed separately and together (dynamic range: 2000–4 pg/ml, 1:2 dilutions, in duplicates), in different stoichiometric ratio. The dilution were done in AlphaLISA NaCl buffer (Perkin Elmer). The protocol is described in the methods sections and called for two incubation times. In (A) the first incubation time lasted 60 min and the second one 30 min, as suggested by the manufacturer’s protocol. In (B), a comparison between different incubation times is shown. The first incubation time was 15, 30, or 60 min, while the second one was 20 min for all the curves. The signal was read on the Perkin Elmer instrument EnVision®.

Article Snippet: The CUSABIO Mouse Pro-Nerve Growth Factor (proNGF) ELISA Kit was purchased and tested.

Techniques: Comparison, Incubation

Journal: Frontiers in molecular neuroscience

Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward.

doi: 10.3389/fnmol.2016.00063

Figure Lengend Snippet: FIGURE 8 | Analysis of transgenic and wild-type mice samples by SPR. Injection of samples from transgenic and from WT mice over the FPro10 anti-proNGF antibody (A) and over the Millipore anti-proNGF antibody (B) (blank subtracted curves). Representation of the SPR curves after subtraction of the WT mice signals. Curve of TgproNGF#72 mice subtracted the curve of WT for the FPro10 anti-proNGF antibody (A—insert), and for the Millipore anti-proNGF antibody (B—insert). In all panels: Red curve: TgproNGF#72, blue curve: WT.

Article Snippet: The CUSABIO Mouse Pro-Nerve Growth Factor (proNGF) ELISA Kit was purchased and tested.

Techniques: Transgenic Assay, Injection

Journal: Frontiers in molecular neuroscience

Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward.

doi: 10.3389/fnmol.2016.00063

Figure Lengend Snippet: FIGURE 1 | IP and WB of transgenic and wild-type mice. (A): IP and WB of cortex extracts from male (M) and female (F) TgProNGF#72 and wild-type (WT) mice. IP on extracts from cortex (CTX) with anti-NGF αD11 antibody, followed by WB with anti-NGF or anti-proNGF antibody, as described in Tiveron et al. (2013). A representative WB probed with anti-proNGF (PAb Alomone), (top) or anti-NGF M20 (Santa Cruz) (bottom) is shown. TgproNGF#72 and wild type mice, male and female, were analyzed. (B) Quantitative analysis of proNGF and mature NGF in the CTX of TgproNGF#72 mice, male and female, by IP and WB and densitometric analysis. After anti-NGF IP, the proNGF bands, (in WB probed with anti-proNGF), and the NGF bands, (in the WB probed with anti-NGF antibody), both identified also by Mass Spectrometry, were quantified. The resulting intensities were normalized against the area of the bands, and then compared with an internal standard of recombinant proNGF and NGF. Loaded samples were in the linear range of detection. Comparison between proNGF and NGF amounts in TgproNGF#72, male and female, is reported in the histogram. The experiment was carried out in triplicate.

Article Snippet: Similarly to what we did for NGF, we tested the only commercially available proNGF ELISA kit (Mouse proNGF ELISA Kit CUSABIO), in order to assess if there is also an interference of NGF on proNGF measurement.

Techniques: Transgenic Assay, Mass Spectrometry, Recombinant, Comparison

Journal: Frontiers in molecular neuroscience

Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward.

doi: 10.3389/fnmol.2016.00063

Figure Lengend Snippet: FIGURE 3 | NGF and proNGF spiked in the sample buffer (supplied by each supplier of the commercial kits). The histograms summarize the results of the NGF and proNGF spiked separately and together into the assay buffer of the three different commercial kits for the detection of NGF analyzed. The Promega kit was tested in the two different formats: rat mAb Promega and αD11. Two concentrations of recombinant NGF (10 and 100 pg/ml) and proNGF (50 and 200 pg/ml) were spiked into the assay buffer either alone or together with NGF and measured by using the kits previously described. (A) (Emax Promega with mAb Promega), (B) (Emax Promega with mAb αD11), (C) (Chemikine Chemicon), (D) (NGF Rapid ELISA Biosensis) report the values of concentration, interpolated by the calibration curves. The calculated values of the samples spiked with both NGF and proNGF were compared to the spiked value of only NGF. The t-student test was carried out and the p-values were calculated. Two asterisks on the histograms mean a p < 0.001.

Article Snippet: Similarly to what we did for NGF, we tested the only commercially available proNGF ELISA kit (Mouse proNGF ELISA Kit CUSABIO), in order to assess if there is also an interference of NGF on proNGF measurement.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Frontiers in molecular neuroscience

Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward.

doi: 10.3389/fnmol.2016.00063

Figure Lengend Snippet: FIGURE 4 | ELISA for the differential detection of NGF and ProNGF. (A) Strategy: capture NGF and measure proNGF (Exploiting the mAb αD11 fast kinetics). ELISA sandwiches format based on a pre-capturing of NGF by a treatment of the sample with mAb αD11 (Cattaneo et al., 1988) on a solid support. Subsequent (Continued)

Article Snippet: Similarly to what we did for NGF, we tested the only commercially available proNGF ELISA kit (Mouse proNGF ELISA Kit CUSABIO), in order to assess if there is also an interference of NGF on proNGF measurement.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Frontiers in molecular neuroscience

Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward.

doi: 10.3389/fnmol.2016.00063

Figure Lengend Snippet: FIGURE 5 | SPR analysis—calibration curves obtained with proNGF and NGF over the panel of different antibodies. (A) Different proNGF concentrations tested on anti-proNGF mAb FPro10. Concentrations in nM (from top): 500, 333, 222, 148, 98, 65, 33, 16, 8, 4, 2, 1, 0.5; (B) Different proNGF concentrations tested on anti-proNGF mAb Millipore. Concentrations in nM (from top): 500, 333, 222, 148, 98, 65, 33, 16, 8, 4, 2, 1, 0.5; (C) Different proNGF concentrations tested on anti-NGF mAb αD11. Concentrations in nM (from top): 500, 333, 222, 148, 98, 65, 33, 16, 8, 4, 2, 1, 0.5; (D) Different proNGF concentrations tested on anti-NGF mAb R&D. Concentrations in nM (from top): 100, 50, 25, 12.50, 6.25, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1; (E) Different NGF concentrations tested on anti-NGF mAb R&D. Concentrations in nM (from top): 100, 50, 25, 12.50, 6.25, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1.

Article Snippet: Similarly to what we did for NGF, we tested the only commercially available proNGF ELISA kit (Mouse proNGF ELISA Kit CUSABIO), in order to assess if there is also an interference of NGF on proNGF measurement.

Techniques:

Journal: Frontiers in molecular neuroscience

Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward.

doi: 10.3389/fnmol.2016.00063

Figure Lengend Snippet: FIGURE 6 | Binding analysis of NGF and proNGF mixtures to different antibodies. The following antibodies were immobilized on the chip for SPR experiments. (A) anti-proNGF mAb FPro10; (B) anti-proNGF mAb Millipore; (C) anti-NGF mAb αD11. In all the panels, the curves represent the following analytes from top to bottom: 200 nM proNGF + 20 nM NGF (solid blue line, experimental value); 200 nM proNGF (solid yellow line, experimental value); 200 nM proNGF + 20 nM NGF (Continued)

Article Snippet: Similarly to what we did for NGF, we tested the only commercially available proNGF ELISA kit (Mouse proNGF ELISA Kit CUSABIO), in order to assess if there is also an interference of NGF on proNGF measurement.

Techniques: Binding Assay

Journal: Frontiers in molecular neuroscience

Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward.

doi: 10.3389/fnmol.2016.00063

Figure Lengend Snippet: FIGURE 7 | AlphaLISA experiments: curves of NGF, proNGF, mix of NGF + proNGF, comparison at different incubation times. Curves of NGF, proNGF, mix of NGF + proNGF, linearly interpolated. NGF and proNGF were assayed separately and together (dynamic range: 2000–4 pg/ml, 1:2 dilutions, in duplicates), in different stoichiometric ratio. The dilution were done in AlphaLISA NaCl buffer (Perkin Elmer). The protocol is described in the methods sections and called for two incubation times. In (A) the first incubation time lasted 60 min and the second one 30 min, as suggested by the manufacturer’s protocol. In (B), a comparison between different incubation times is shown. The first incubation time was 15, 30, or 60 min, while the second one was 20 min for all the curves. The signal was read on the Perkin Elmer instrument EnVision®.

Article Snippet: Similarly to what we did for NGF, we tested the only commercially available proNGF ELISA kit (Mouse proNGF ELISA Kit CUSABIO), in order to assess if there is also an interference of NGF on proNGF measurement.

Techniques: Comparison, Incubation

Journal: Frontiers in molecular neuroscience

Article Title: NGF and proNGF Reciprocal Interference in Immunoassays: Open Questions, Criticalities, and Ways Forward.

doi: 10.3389/fnmol.2016.00063

Figure Lengend Snippet: FIGURE 8 | Analysis of transgenic and wild-type mice samples by SPR. Injection of samples from transgenic and from WT mice over the FPro10 anti-proNGF antibody (A) and over the Millipore anti-proNGF antibody (B) (blank subtracted curves). Representation of the SPR curves after subtraction of the WT mice signals. Curve of TgproNGF#72 mice subtracted the curve of WT for the FPro10 anti-proNGF antibody (A—insert), and for the Millipore anti-proNGF antibody (B—insert). In all panels: Red curve: TgproNGF#72, blue curve: WT.

Article Snippet: Similarly to what we did for NGF, we tested the only commercially available proNGF ELISA kit (Mouse proNGF ELISA Kit CUSABIO), in order to assess if there is also an interference of NGF on proNGF measurement.

Techniques: Transgenic Assay, Injection