grk inhibitor  (Santa Cruz Biotechnology)

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Phosphoproteomic and Functional Analyses Reveal Sperm-specific Protein Changes Downstream of Kappa Opioid Receptor in Human Spermatozoa *

doi: 10.1074/mcp.RA118.001133

Figure Lengend Snippet: Proteomic and phosphoproteomic analysis of human spermatozoa. A,, Schematic representation of the experimental workflow followed in the study. Three biological replicas of each untreated (Ctrl) and treated spermatozoa (U50488H) were used for 1 and 60 min. For both time point, after the protein extraction of each sample, the soluble and insoluble protein fractions were separated and independently subjected to a in-solution digestion. To analyze the whole proteome in human spermatozoa, 1/10 of each sample were used for tandem mass tag (TMT-6plex) labeling and after performing a high pH fractionation, the peptide mixture was analyzed by LC-MS/MS. On the other hand, the other 9/10 of each sample was utilized for TiO2-based phospho-enrichment followed by the subsequent TMT labeling and LC-MS/MS analysis. This procedure was followed independently with both soluble and insoluble protein fractions treated with U50488H for 1 and 60 min. B,, Gene ontology analysis (PANTHER bioinformatic tool) indicating the biological processes of the total proteins identified in the study. The fold enrichment and the statistical significance p, value of the most indicative terms are indicated. The size of the dots correlate with the number of proteins grouped in the same term. C,, Total Class I phosphosite (p-sites) and corresponding proteins quantified in the three biological replicas. Distribution of all quantified phosphorylated serine, threonine and tyrosine residues. D,, Gene ontology analysis (PANTHER bioinformatic tool) indicating the biological processes of the proteins belonging to the Class I site phosphosites. The fold enrichment and the statistical significance p, value of the most indicative terms are indicated. E,, Venn diagrams comparing the number of the identified proteins (left side) and phosphosites (right side) in this study with the published studies.

Article Snippet: For functional analyses the samples were co- incubated with U50488H (Sigma-Aldrich) and the different activators and inhibitors of different signaling pathways.

Techniques: Protein Extraction, Labeling, Fractionation, Liquid Chromatography with Mass Spectroscopy

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Phosphoproteomic and Functional Analyses Reveal Sperm-specific Protein Changes Downstream of Kappa Opioid Receptor in Human Spermatozoa *

doi: 10.1074/mcp.RA118.001133

Figure Lengend Snippet: Effect of U50488H in the human sperm phosphoproteome. A,, Overall Log2 U50488H/Control TMT fold change as a function of -Log2 statistical significance of U50488H/Control (p, value < 0.05) of the soluble and insoluble protein fractions after 1 and 60 min U50488H treatment. In blue are indicated the down-regulated phosphosites by U50488H. In red are represented the U50488H-up-regulated phosphosites. B,, Tables containing the U50488H-regulated phosphosites following 1 and 60 min treatment in the soluble and insoluble protein fractions. U50488H/Ctrl > 1.5 in red and U50488H/Ctrl < 0.67 in blue. C,, Representation of a working model of the protein interactions belonging to the U50488H-regulated phosphosites. KOR is represented in green color whereas the proteins belonging to the U50488H-regulated phosphosites are colored in orange. The showing model has been constructed based on GENEMANIA (http://genemania.org/) the interactive functional association network tool. D,, Illustration gathering the localization of the proteins belonging to the U50488H-regulated phosphosites within the human spermatozoa. The downregulated phoshosites are represented in blue whereas the up-regulated ones are colorized in red.

Article Snippet: For functional analyses the samples were co- incubated with U50488H (Sigma-Aldrich) and the different activators and inhibitors of different signaling pathways.

Techniques: Construct, Functional Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Phosphoproteomic and Functional Analyses Reveal Sperm-specific Protein Changes Downstream of Kappa Opioid Receptor in Human Spermatozoa *

doi: 10.1074/mcp.RA118.001133

Figure Lengend Snippet: Role of kappa-opioid receptor in human sperm physiology. Effect of 1 and 60 min U50488H (1 μm) (Kappa-opioid receptor agonist) treatments in human sperm motility, capacitation and acrosome reaction. A,, Immunoblotting assays showing the expression of the phosphorylated substrates of protein kinase C following 1 and 60 min U50488H treatment. n, = 3. (**p, < 0.01 versus, Control). B,, Motility assays using the CASA (Computer Assisted Sperm Analyzer) system for the study of 1 and 60 minutes U50488H treatment in the progressive, nonprogressive, immotile and hyperactive motilities. X axis shows the motility types measured in the study and the Y axis represents the normalized data of the % of motile spermatozoa. The normalization was performed using the untreated samples (Control). n, = 8. (*p, < 0.05 and **p, < 0.01 versus, Control). C,, Immunoblotting assays for the study of U50488H in human sperm capacitation using the anti-pTyr antibody. D,, Flow citometry experiments for the study of U50488H in the human sperm acrosome reaction. X axis shows the different treatments used in the study and the Y axis represents the normalized data of the % acrosome reacted spermatozoa. The normalization was performed using the untreated samples and the acrosome reacted samples. The progesterone was used as the positive control of the acrosome reaction. n, = 7. (**p, < 0.01 versus, Control) AR: acrosome reaction. P-subtrates PKC: Phosphorylated substrates of PKC.

Article Snippet: For functional analyses the samples were co- incubated with U50488H (Sigma-Aldrich) and the different activators and inhibitors of different signaling pathways.

Techniques: Western Blot, Expressing, Positive Control

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Phosphoproteomic and Functional Analyses Reveal Sperm-specific Protein Changes Downstream of Kappa Opioid Receptor in Human Spermatozoa *

doi: 10.1074/mcp.RA118.001133

Figure Lengend Snippet: Study of the hyperactive motility and acrosome reaction calcium downstream KOR in human spermatozoa. A,, Study of the hyperactive motility by 1 min co-incubation of U50488H (1 μm) and: NNC-55–0395 (10 μm), the Catsper inhibitor; Mibefradil (30 μm), a calcium channel activator and U73122 (3 μm), the PLC inhibitor. X axis shows the different treatments used for this study and the Y axis represents the normalized data of the % of motile spermatozoa. The normalization was performed using the untreated samples. n, = 6. (*p, < 0.05 and **p, < 0.01 versus, Control; +p, < 0.05 versus, Mibefradil). B,, Study of the acrosome reaction by the 60 min co-incubation of U50488H (1 μm) and: NNC-55–0395 (10 μm), Mibefradil (30 μm) and U733122 (3 μm). X axis shows the different treatments used for this study and the Y axis represents the normalized data of the % of acrosome reacted spermatozoa. The normalization was performed using the untreated samples and the acrosome reacted samples. The progesterone was used as the positive control of the acrosome reaction. n, = 7. (*p, < 0.05 and **p, < 0.01 versus, Control; +p, < 0.05 versus, Mibefradil). C,, Intracellular free Ca2+ measurements in human sperm cells loaded with Fura-2 in response to the solvent of U50488H (H2O) (control samples) and U50488H (1 μm). Subsequent addition of 30 μm Mibefradil as a calcium channel activator and 1 μm progesterone to the same sperm aliquots. The progesterone caused a typical biphasic [Ca2+]i progesterone response. The X axis shows time in seconds and the Y axis shows [Ca2+]i expressed by Fura-2 ratio variation. Calibration of [Ca2+]i was achieved adding Triton X-100 (TX), to obtain the maximal response, followed by addition of EGTA to obtain the minimal response. n, = 5. AR = Acrosome reacted. HA = Hyperactive.

Article Snippet: For functional analyses the samples were co- incubated with U50488H (Sigma-Aldrich) and the different activators and inhibitors of different signaling pathways.

Techniques: Incubation, Positive Control