At 3 μM, PF-04822163 has little effect on other cyclic nucleotide phosphodiesterases except for PDE10 43 , which is not expressed at a detectable level ( Supplemental Figs. 5, B and C ). We also attempted to inhibit protein kinase A using Rp-cAMPS, Rp-8-Br-cAMPS, and Rp-8-CPT-cAMPS, but these were ineffective, as indicated by the failure to block phosphorylation of the protein kinase A substrate VASP, perhaps due to insufficient permeability ( Supplemental Fig. B shows a blot of follicle protein separated in a gel without Phos-tag, and probed with the pS92-PDE5 antibody (representative of four experiments).
B, C) Forskolin (100 μM, 30 min) caused an increase in PDE5 phosphorylation similar to that seen with LH, and H89 (100 μM, 2 h pre-incubation) inhibited PDE5 phosphorylation in response to LH or forskolin. However, only a low level of PDE5 phosphorylation was seen prior to LH exposure ( Figs. Proteins were separated in a Phos-tag-containing gel, and the blot was probed with a total PDE5 antibody.E) Time course of the LH-induced increase in PDE5 phosphorylation, summarizing the results of three blots like that shown in D. The graph shows ratios of the immunodensity of the upper band divided by that of the lower band. In D, proteins were separated in a Phos-tag-containing gel, and the blot was probed with a total PDE5 antibody (representative of three experiments). C, D) Western blots showing the time course of PDE5 phosphorylation in follicles treated for 10-240 min with LH. In C, proteins were separated in a gel without Phos-tag, and the blot was probed with the pS92-PDE5 antibody (representative of three experiments).Proteins were separated in a Phos-tag-containing gel, and blots were probed with antibodies recognizing total PDE5 (left) or pS92-PDE5 (right). B) Western blots showing the increase in PDE5 phosphorylation in follicles treated with LH for 30 min. PDE5 phosphorylation on serine 92 increases in response to LH. A) Functional domains of PDE5 showing the phosphorylation site.4A ), we separated follicle proteins in a Phos-tag-containing gel and probed a blot of the gel with an antibody specific for PDE5-phospho-serine 92 (pS92). LH Increases Phosphorylation of PDE5 on Its Regulatory Serine. Studies to investigate the basal level of free Ca2+ in the granulosa cells, and to determine whether and to what extent LH signaling increases free Ca2+, would contribute to understanding of the function of PDE1 in the follicle.Because Ca2+/calmodulin does not stably modify PDE1, our measurements of PDE1 activity in follicle lysates do not provide information as to whether LH signaling increases Ca2+. These results confirm that the PF-04822163-sensitive activity is indeed due to PDE1 and that if LH signaling increases Ca2+, PDE1 activity would increase. Activity values obtained in the presence of Ca2+ and calmodulin were ∼6-fold those obtained in the presence of EGTA ( Fig.Because PDE1 activity is increased by binding of Ca2+/calmodulin and because some evidence indicates that LH signaling might elevate Ca2+ in the follicle (see Discussion), we compared PDE1 activity in follicle lysates with and without the addition of CaCl2 (20-80 μM free Ca2+) and calmodulin.https://cenforces.com/https://strongtabs.com/
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