primary antibodies against gnrhr  (Proteintech)

Journal: International Journal of Nanomedicine

Article Title: Pseudoephedrine Nanoparticles Alleviate Adriamycin-Induced Reproductive Toxicity Through the GnRhR Signaling Pathway

doi: 10.2147/ijn.s348673

Figure Lengend Snippet: Figure 4 Effect of PSE-NPs on the GnRhR signaling pathway and expression of meiosis-related proteins SCP3 of GC-1 cells damaged by adriamycin: (A–E) Protein expression of GnRhR, GNAS, p-CREB and SCP3 in GC-1 cells, scale bar 50 μm. Notes: Data are the mean ± SD (n = 6). *P < 0.05 and **P < 0.01 vs the CON group. ##P < 0.01 vs the Model group. &&P < 0.01 vs the PSE group. Abbreviations: CON, control group underwent standard culture; M, model group; PLGA, empty nanoparticles; PSE, 0.5μM pseudoephedrine; PSE-PLGA, 0.25 μM pseudoephedrine-loaded PLGA nanoparticles; GnRhR, gonadotropin-releasing hormone receptor; CREB, cyclic adenosine monophosphate (cAMP)-response element binding protein; GNAS, guanine nucleotide binding protein; SCP3, small C-terminal domain phosphatase 3.

Article Snippet: After membranes were blocked with 5% BSA in buffer for 2 h, primary antibodies against GnRhR (1:500 dilution; 19950-1-AP; Proteintech), guanine nucleotide binding protein (GNAS; 1:1000; ab283266; Abcam), protein kinase A (PKA; 1:1000; 12232-1-AP, Proteintech), phosphorylated-cyclic adenosine monophosphate (cAMP)-response element binding protein (p-CREB; 1:1000; 9198; Cell Signaling Technology, Danvers, MA, USA), CREB (1:1000; 12208-1-AP; Proteintech), SCP3 (small C-terminal domain phosphatase 3, 1:1000; 23024- 1-AP; Proteintech), REC8 meiotic recombination protein (REC8; 1:1000; ab192241; Proteintech), cAMP (Cyclic Adenosine monophosphate, 1:1000; ab134901; Abcam), Caspase 3 (1:1000; ab32351, Abcam), Bax (BCL2-Associated X, 1:1000; 60267-1-Ig, Proteintech), Bcl-2 (B cell lymphoma-2, 1:1000; 60178-1-Ig, Proteintech) or β-Actin (1:5000; 66009-1-Ig; Proteintech) were added and incubation allowed to occur overnight at 4°C.

Techniques: Expressing, Control, Binding Assay

Journal: International Journal of Nanomedicine

Article Title: Pseudoephedrine Nanoparticles Alleviate Adriamycin-Induced Reproductive Toxicity Through the GnRhR Signaling Pathway

doi: 10.2147/ijn.s348673

Figure Lengend Snippet: Figure 7 Effects of PSE-NPs on sex-hormone levels, GnRh signaling pathway-related proteins in testes, and indices of meiosis in mice: (A) Levels of GnRh, LH and testosterone in the serum of mice in each group. (B) mRNA expression of GnRh signaling pathway-related proteins and meiosis-associated genes in the testicular tissue of mice in each group was measured by RT-qPCR. (C) Protein expression of GnRh signaling pathway and meiosis-associated proteins in the testicular tissue of mice in each group was measured by western blotting. (D) The protein expression of GnRhR and SCP3 in the testicular tissue of mice in each group was measured by Immunofluorescence, scale bar 50 μm. Notes: Data are the mean ± SD (n = 6). *P < 0.05 and **P < 0.01 vs the CON group. #P < 0.05 and ##P < 0.01 vs the Model group. &P < 0.05 and &&P < 0.01 vs the PSE group. Abbreviations: CON, Control group; M, Model group with 30 mg/kg adriamycin, i.p.; PSE, 1.4 mg/kg PSE, i.v.; PLGA, 53.8 mg/kg PLGAs, i.v.; PSE-PLGA, 53.8 mg/kg PSE- PLGAs, i.v.; GnRh, gonadotropin-releasing hormone; LH, luteinizing hormone; GnRHR, gonadotropin-releasing hormone receptor; LHR, luteinising hormone receptor; AR, androgen receptor; GNAS, guanine nucleotide-binding proteins; ADCY1, adenylate cyclase type 1; PKA, protein kinase A; DAZ1, deleted in azoospermia protein 1; DDX4, Dead box polypeptide 4; STAR, StAR-related lipid transfer protein 8; SCP3, synaptonemal complex protein 3; REC8, meiotic recombination protein; SMC1B, structural maintenance of chromosomes protein 1B; Miwi, murine piwi gene; cAMP, Cyclic Adenosine monophosphate.

Article Snippet: After membranes were blocked with 5% BSA in buffer for 2 h, primary antibodies against GnRhR (1:500 dilution; 19950-1-AP; Proteintech), guanine nucleotide binding protein (GNAS; 1:1000; ab283266; Abcam), protein kinase A (PKA; 1:1000; 12232-1-AP, Proteintech), phosphorylated-cyclic adenosine monophosphate (cAMP)-response element binding protein (p-CREB; 1:1000; 9198; Cell Signaling Technology, Danvers, MA, USA), CREB (1:1000; 12208-1-AP; Proteintech), SCP3 (small C-terminal domain phosphatase 3, 1:1000; 23024- 1-AP; Proteintech), REC8 meiotic recombination protein (REC8; 1:1000; ab192241; Proteintech), cAMP (Cyclic Adenosine monophosphate, 1:1000; ab134901; Abcam), Caspase 3 (1:1000; ab32351, Abcam), Bax (BCL2-Associated X, 1:1000; 60267-1-Ig, Proteintech), Bcl-2 (B cell lymphoma-2, 1:1000; 60178-1-Ig, Proteintech) or β-Actin (1:5000; 66009-1-Ig; Proteintech) were added and incubation allowed to occur overnight at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Control, Binding Assay

Journal: Bioconjugate chemistry

Article Title: GnRH-Gemcitabine conjugates for the treatment of androgen-independent prostate cancer: pharmacokinetic enhancements combined with targeted drug delivery.

doi: 10.1021/bc500081g

Figure Lengend Snippet: Figure 1. Design of GnRH−gemcitabine conjugates for selective delivery to GnRH-R positive cancer cells. A. Representative positive electrospray ionization (ESI+) mass spectra of GnRH-gemcitabine conjugates showing the three ionized forms (m/z 1599.3 corresponding to M+1, m/z 800.7 corresponding to M+2, m/z 534.1 corresponding to M+3) of GSG. B. LC-MS/MS chromatogram depicts the separation of GSG, gemcitabine and dFdU.

Article Snippet: The membranes were then blocked in 5% nonfat dry milk in TBS-T buffer for 1 h at room temperature and were then incubated overnight at 4 °C with 1:500 dilution of rabbit polyclonal primary antibody against GnRH-R1 (Proteintech, UK).

Techniques: Liquid Chromatography with Mass Spectroscopy

Journal: Bioconjugate chemistry

Article Title: GnRH-Gemcitabine conjugates for the treatment of androgen-independent prostate cancer: pharmacokinetic enhancements combined with targeted drug delivery.

doi: 10.1021/bc500081g

Figure Lengend Snippet: Figure 2. Evaluation of the binding potential of representative GnRH−gemcitabine conjugates against the GnRH-R using 2D NMR and radioligand binding assays. A. Schematic representation of a GnRH−gemcitabine conjugate indicating the residues that are responsible for binding to the GnRH- R. B. Superimposition of the selected region NH−of 2D NMR TOCSY spectrum of [D-Lys6]-GnRH (black) with 3G (red) and GSG (blue). The conjugated molecules, GSG and 3G, do not perturb the structure of the [D-Lys6]-GnRH region that is important for binding to the GnRH-R. C. Binding affinity (IC50) of GnRH−gemcitabine conjugates (3G, GSG, 3G2, GSG2) on the GnRH-R was evaluated in comparison to the binding affinity of the known GnRH-R superagonist leuprolide as well as the binding affinity of ([D-Lys6]-GnRH). Competition binding isotherms of GnRH analogues to human GnRH −I receptor are also shown. Competition of [125I-D-Tyr6, His5] GnRH specific binding by increasing concentrations of the analogues was performed on membranes from HEK 293 cells stably expressing the human GnRH −I receptor. The mean values and SE are shown from 3−4 different experiments. The data were fit to a one-site competition model by nonlinear regression and the IC50 values were determined as described.

Article Snippet: The membranes were then blocked in 5% nonfat dry milk in TBS-T buffer for 1 h at room temperature and were then incubated overnight at 4 °C with 1:500 dilution of rabbit polyclonal primary antibody against GnRH-R1 (Proteintech, UK).

Techniques: Binding Assay, Comparison, Analogues, Stable Transfection, Expressing

Journal: Bioconjugate chemistry

Article Title: GnRH-Gemcitabine conjugates for the treatment of androgen-independent prostate cancer: pharmacokinetic enhancements combined with targeted drug delivery.

doi: 10.1021/bc500081g

Figure Lengend Snippet: Figure 3. Pharmacokinetic evaluation of GnRH−gemcitabine conjugates versus gemcitabine. Male C57BL/6N mice (n = 5) were dosed (IP) with either gemcitabine, GSG, or 3G2 at a dose of 6.3 μmol/kg, and blood samples were collected at selected time points. Gemcitabine and dFdU were monitored by LC-MS/MS. The areas under the curve (AUCs) for each treatment were calculated as a measure of gemcitabine or dFdU exposure over time.

Article Snippet: The membranes were then blocked in 5% nonfat dry milk in TBS-T buffer for 1 h at room temperature and were then incubated overnight at 4 °C with 1:500 dilution of rabbit polyclonal primary antibody against GnRH-R1 (Proteintech, UK).

Techniques: Liquid Chromatography with Mass Spectroscopy

Journal: Bioconjugate chemistry

Article Title: GnRH-Gemcitabine conjugates for the treatment of androgen-independent prostate cancer: pharmacokinetic enhancements combined with targeted drug delivery.

doi: 10.1021/bc500081g

Figure Lengend Snippet: Figure 4. Cell culture GnRH-R expression and cell uptake of a GnRH−gemcitabine conjugate. A. GnRH-R expression in the two androgen-independent cell lines used (DU145, PC3) for the evaluation of the antiproliferative effects of the GnRH−gemcitabine conjugates. MCF-7 and RAW264.7 cells were used as positive (+) and negative (-) controls, respectively. Expression of the GnRH-R in androgen-independent cell lines was confirmed by Western blot analysis as a band at 38 kDa. B. GSG vs gemcitabine cell uptake in DU145 cells. Cells were incubated with 10 μM GSG or gemcitabine for selected time points (1 h, 4 h, 8 h) and were then lysed in order to determine intracellular levels of gemcitabine and its inactive metabolite (dFdU) by LC-MS/MS. Experiments were performed in triplicate.

Article Snippet: The membranes were then blocked in 5% nonfat dry milk in TBS-T buffer for 1 h at room temperature and were then incubated overnight at 4 °C with 1:500 dilution of rabbit polyclonal primary antibody against GnRH-R1 (Proteintech, UK).

Techniques: Cell Culture, Expressing, Western Blot, Incubation, Liquid Chromatography with Mass Spectroscopy

Journal: Bioconjugate chemistry

Article Title: GnRH-Gemcitabine conjugates for the treatment of androgen-independent prostate cancer: pharmacokinetic enhancements combined with targeted drug delivery.

doi: 10.1021/bc500081g

Figure Lengend Snippet: Figure 5. Therapeutic efficacy of GSG in NOD/SCID mice xenografted with DU145 cells. A. Tumor growth inhibition. Mice were dosed (IP) with GSG, [D-Lys6]-GnRH, gemcitabine (low and high dose), or vehicle (saline). Red arrows indicate the day of dosing. Each point represents the mean of at least 8 tumor volumes resulting from at least 5 mice ± SD ***, P < 0.001 vs controls. B. AUC (mm3 . day) calculated for each treatment group from the tumor volumes of mice.

Article Snippet: The membranes were then blocked in 5% nonfat dry milk in TBS-T buffer for 1 h at room temperature and were then incubated overnight at 4 °C with 1:500 dilution of rabbit polyclonal primary antibody against GnRH-R1 (Proteintech, UK).

Techniques: Inhibition, Saline