recombinant human pde8a  (Millipore)

Journal: bioRxiv

Article Title: Regulation of intracellular cAMP levels in osteocytes by mechano-sensitive focal adhesion kinase via PDE8A

doi: 10.1101/2024.06.28.601153

Figure Lengend Snippet: Gsα, PTHR1 and PDE8A tyrosine phosphorylation sites identified by phosphoproteomics to be reduced by FAK inhibition or deletion.

Article Snippet: Recombinant human PDE8A (Sigma SRP0273) was incubated with 100 ng FAK tyrosine kinase (Promega #V1971) 50μM DTT, 2mM MnCl 2 , 50μM ATP and 1x kinase buffer for 15 min at 37°C in a reaction of total volume 25 µL.

Techniques: Inhibition

Journal: bioRxiv

Article Title: Regulation of intracellular cAMP levels in osteocytes by mechano-sensitive focal adhesion kinase via PDE8A

doi: 10.1101/2024.06.28.601153

Figure Lengend Snippet: ( a ) Left: GloSensor cAMP assay in Saos2 cells following PDE8A inhibitor treatment (PF04957325, 10 μM). Right: Dose response of the PDE8A inhibitor PF04957325 in cAMP in GloSensor- expressing Saos2 cells. cAMP luminescence over time is shown. ( b ) Left: PDE8A immunoblotting in PDE8A KO cells. Right : GloSensor assay revealed increased cAMP luminescence in PDE8A KO cells compared to control cells upon treatment with isoproterenol (10 -6 M). Control cells were transfected with the empty vector PX459. ( c ) qRT-PCR showing relative expression of Sost ± treatment with PDE8A inhibitor 10 μM (left) and dose response of PDE8A inhibitor (right). Cells were treated for 4 hours prior to RNA isolation. For each group in control vs PDE8A inhibitor treated cells n = 6 biologic replicates for RNA and two-sided unpaired t test were used. N=2 biologic replicates were used for the dose response experiment.

Article Snippet: Recombinant human PDE8A (Sigma SRP0273) was incubated with 100 ng FAK tyrosine kinase (Promega #V1971) 50μM DTT, 2mM MnCl 2 , 50μM ATP and 1x kinase buffer for 15 min at 37°C in a reaction of total volume 25 µL.

Techniques: cAMP Assay, Expressing, Western Blot, Control, Transfection, Plasmid Preparation, Quantitative RT-PCR, Isolation

Journal: bioRxiv

Article Title: Regulation of intracellular cAMP levels in osteocytes by mechano-sensitive focal adhesion kinase via PDE8A

doi: 10.1101/2024.06.28.601153

Figure Lengend Snippet: ( a ) Immunoblotting for phospho-tyrosine (pY) and PDE8A in vitro FAK kinase assay with recombinant human PDE8A. pY bands are located at the expected size of FAK (auto-phosphorylation) and PDE8A. ( b ) Immunoblotting for FLAG and V5 in lysates from cells transfected with plasmids expressing FAK and PDE8A tagged with FLAG and V5 respectively after immunoprecipitation with anti-FLAG affinity gel. ( c ) Immunoblotting for FLAG and V5 in protein lysates from cells transfected with plasmids expressing FAK and PDE8A tagged with FLAG and V5 respectively after immunoprecipitation with anti-V5 affinity gel.

Article Snippet: Recombinant human PDE8A (Sigma SRP0273) was incubated with 100 ng FAK tyrosine kinase (Promega #V1971) 50μM DTT, 2mM MnCl 2 , 50μM ATP and 1x kinase buffer for 15 min at 37°C in a reaction of total volume 25 µL.

Techniques: Western Blot, In Vitro, Kinase Assay, Recombinant, Transfection, Expressing, Immunoprecipitation

Journal: bioRxiv

Article Title: Regulation of intracellular cAMP levels in osteocytes by mechano-sensitive focal adhesion kinase via PDE8A

doi: 10.1101/2024.06.28.601153

Figure Lengend Snippet: ( a ) PDE8A dose response. X axis: recombinant PDE8A dose in ng/μl y axis: 665/620 TR-FRET signal ratio (inversely proportional to cAMP levels) ( b ) 665/620 ratio after incubation of recombinant PDE8A with different amounts of recombinant FAK (shown on axis x). All reactions occurred in presence of 6 nM cAMP. TR-FRET signal at 665 nm was normalized to the signal of the donor-channel at 620 nm to correct for well-to-well variability of the signal

Article Snippet: Recombinant human PDE8A (Sigma SRP0273) was incubated with 100 ng FAK tyrosine kinase (Promega #V1971) 50μM DTT, 2mM MnCl 2 , 50μM ATP and 1x kinase buffer for 15 min at 37°C in a reaction of total volume 25 µL.

Techniques: Recombinant, Incubation

Journal: bioRxiv

Article Title: Regulation of intracellular cAMP levels in osteocytes by mechano-sensitive focal adhesion kinase via PDE8A

doi: 10.1101/2024.06.28.601153

Figure Lengend Snippet: Normally, active FAK phosphorylates PDE8A and maintains high basal PDE8A-mediated cAMP hydrolysis. FFSS causes a reduction of FAK activity leading to reduced FAK-mediated PDE8A phosphorylation and, in turn, reduced PDE8A-mediated cAMP breakdown. Thus, FAK inhibition leads to suppressed PDE8A function causing an accumulation of cAMP that is generated by upstream GPCR signaling.

Article Snippet: Recombinant human PDE8A (Sigma SRP0273) was incubated with 100 ng FAK tyrosine kinase (Promega #V1971) 50μM DTT, 2mM MnCl 2 , 50μM ATP and 1x kinase buffer for 15 min at 37°C in a reaction of total volume 25 µL.

Techniques: Activity Assay, Inhibition, Generated