Journal: Biology of Reproduction

Article Title: Characterization of Novel Phosphodiesterases in the Bovine Ovarian Follicle 1

doi: 10.1095/biolreprod.108.074450

Figure Lengend Snippet: Immunoblot detection of PDE8A, PDE8B, PDE10A, PDE4D, and α-tubulin in granulosa cells and COCs. A) The expression of various PDEs was determined by immunodetection. Protein samples were run on SDS-PAGE and hybridized with specific PDE antibodies. For each gel, a molecular mass ladder was run with the samples, and band positions are displayed on the left in kiloDaltons. Samples are rat brain (RB), mouse ovary (MO), bovine testis (BT), mural granulosa cells (MGC) from 2- to 6-mm follicles, and COCs. B) Comparative expression of PDE8A between granulosa cells from 2- to 6-mm follicles and follicles larger than 10 mm. PDE8A and α-tubulin intensities were quantified by ImageJ software (National Institutes of Health) from two separate experiments. The intensity ratio presented below the figure is expressed as the mean ratio between PDE8A and α-tubulin.

Article Snippet: Membranes were blotted with the primary antibody PDE8A (diluted 1:500; FabGennix International Inc., Frisco, TX) at room temperature for 2 h, after which the membranes were exposed to goat anti-rabbit peroxidase-conjugated secondary antibody (diluted 1:40 000; Jackson Immunoresearch Laboratories Inc., Bar Harbor, ME).

Techniques: Western Blot, Expressing, Immunodetection, SDS Page, Software

Journal: Biology of Reproduction

Article Title: Characterization of Novel Phosphodiesterases in the Bovine Ovarian Follicle 1

doi: 10.1095/biolreprod.108.074450

Figure Lengend Snippet: Detection of PDE3A, PDE8A, PDE8B, PDE10A, PDE11A, and ACTB expression by RT-PCR in the various compartments of the ovarian follicle. For each gel, a molecular mass marker (M) was run with the samples, with the corresponding molecular weight (in base pairs) noted on the left. PDE genes were amplified from oocyte (Oo), cumulus (Cc), and granulosa cell (Gc) cDNA, along with a no-template negative control (−) and a mouse mammary gland positive control (+). A representative experiment of three replicates is shown.

Article Snippet: Membranes were blotted with the primary antibody PDE8A (diluted 1:500; FabGennix International Inc., Frisco, TX) at room temperature for 2 h, after which the membranes were exposed to goat anti-rabbit peroxidase-conjugated secondary antibody (diluted 1:40 000; Jackson Immunoresearch Laboratories Inc., Bar Harbor, ME).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Marker, Molecular Weight, Amplification, Negative Control, Positive Control

Journal: International Journal of Molecular Sciences

Article Title: Phosphodiesterases Expression during Murine Cardiac Development

doi: 10.3390/ijms22052593

Figure Lengend Snippet: Overview of PDE isoforms involved in cardiac functions.

Article Snippet: The membrane was immersed in 5% v / v blocking milk (TBST + 5% non-fat milk) for 1 h at room temperature and incubated in primary antibodies (1:500 v / v in TBST + 5% w / v BSA (bovine serum albumin) buffer) for phosphodiesterase detection (PDE1A FabGennix #PDE1A-101AP; PDE1C FabGennix #PD1C301AP; PDE2A Abcam #ab140650; PDE3A Santa Cruz #sc-20792; PDE4A Proteintech #16226-1-AP; PDE4D Invitrogen #PA5-79795; PDE5A Santa Cruz #32884; PDE8A Sigma Aldrich #HPA007722) overnight at 4 °C.

Techniques: Transgenic Assay, Knock-Out, Over Expression

Journal: Frontiers in Endocrinology

Article Title: cAMP-specific phosphodiesterase 8A and 8B isoforms are differentially expressed in human testis and Leydig cell tumor

doi: 10.3389/fendo.2022.1010924

Figure Lengend Snippet: PDEs expression analysis in human testis (A) PDEs mRNA expression analysis by QuantiGene Bioplex Assay on human testis biopsies from healthy subjects. Results, normalized for HPRT1, are expressed as arbitrary units (A.U, (median and 5-95 percentile) and reported in the histogram bar chart (n=4). (B) PDE8 isoforms expression analysis by immunohistochemistry using commercially available anti-PDE8A and anti-PDE8B antibodies on human testis biopsies. Scale bars represent 20 μm. (C) Localization of PDE8 isoforms by immunofluorescence analysis on sections obtained from human testis biopsies of healthy subjects. PDE8 isoforms staining is shown in green and nuclei are shown in blue (DAPI). Scale bars represent 20 μm except for the insets where they represent 10 μm.

Article Snippet: Sections were incubated with primary antibodies overnight at 4°C: anti-PDE8A (Atlas Antibodies Cat#HPA007722, RRID : AB_1855130), PDE8B (Atlas Antibodies Cat#HPA036912, RRID : AB_10670377), anti-ACROSIN (Biosonda, Santiago, Chile; Cat No. AMC-ACRO-C5F10-AS), Anti-Golgin-97 (SC-59820, Santa Cruz Biotechnology), anti-GM130 (610822 BD Biosciences), Lectin-PNA Alexa Fluor 488 (L21409 ThermoFisher Scientific).

Techniques: Expressing, BioPlex Assay, Immunohistochemistry, Immunofluorescence, Staining

Journal: Frontiers in Endocrinology

Article Title: cAMP-specific phosphodiesterase 8A and 8B isoforms are differentially expressed in human testis and Leydig cell tumor

doi: 10.3389/fendo.2022.1010924

Figure Lengend Snippet: PDE8 expression analysis in LCTs (A) PDE8A and PDE8B mRNA expression analysis by QuantiGene Bioplex Assay on human testis biopsies from healthy subjects (NT) and LCT patients. Results, normalized for HPRT1, are expressed as arbitrary units (A.U, (median and 5-95 percentile) and reported in the histogram bar chart (n=4). (B) PDE8 isoforms mRNA expression analysis by qPCR, normalized for HPRT1, on human testis biopsies from healthy subjects (NT) and LCT patients. Results are reported as fold increase vs NT (mean ± SEM, n=9) in the histogram bar chart. *p<0.05. (C) PDE8 isoforms expression analysis by immunohistochemistry using commercially available anti-PDE8A and PDE8B antibodies on normal testis biopses (NT) and LCTs. Negative control (NC) is represented in the right panels. Scale bar are depicted beneath the panels.

Article Snippet: Sections were incubated with primary antibodies overnight at 4°C: anti-PDE8A (Atlas Antibodies Cat#HPA007722, RRID : AB_1855130), PDE8B (Atlas Antibodies Cat#HPA036912, RRID : AB_10670377), anti-ACROSIN (Biosonda, Santiago, Chile; Cat No. AMC-ACRO-C5F10-AS), Anti-Golgin-97 (SC-59820, Santa Cruz Biotechnology), anti-GM130 (610822 BD Biosciences), Lectin-PNA Alexa Fluor 488 (L21409 ThermoFisher Scientific).

Techniques: Expressing, BioPlex Assay, Immunohistochemistry, Negative Control

Journal: Scientific Reports

Article Title: Disruption of the pro-oncogenic c-RAF–PDE8A complex represents a differentiated approach to treating KRAS–c-RAF dependent PDAC

doi: 10.1038/s41598-024-59451-3

Figure Lengend Snippet: In vitro DRx-170 sensitivity vs. PANC1 and Panc08.13 human PDAC cell lines. ( A ) Scatter plot highlighting KRAS and c-RAF gene effect (RNAi, Achilles + DRIVE + Marcotte, DEMETER2) in 28 pancreatic adenocarcinoma (PAAD) human cancer cell lines (DepMap). See Supplementary Data Set . ( B ) RTCA (xCELLigence) of (i) PANC1 and (ii) Panc08.13 following 48 h treatment with vehicle [1% DMSO] or DRx-170 [0.1 µM]. (iii) Representative bar chart of relative rate of growth (MEAN ± SEM, n = 6). ( C ) Representative immunoblots of KRAS, c-RAF, Total Protein (Revert ™ 700 nm stain) from PANC1 and Panc08.13. Corresponding bar chart of relative protein expression (N = 4, ns; not significant, two-way ANOVA). ( D ) DepMap derived Reverse Phase Protein Array (RPPA) data of relative Y1068 EGFR, Y1173 EGFR, Y1248 ERBB2, Y1289 ERBB3, pS299 A-RAF, pS445 B-RAF, pS338 c-RAF, S217/S221 MEK1/2, T202/Y204 ERK1/2 phosphorylated protein expression in PANC1 and Panc08.13 (Log2). See Supplementary Data Set .

Article Snippet: Primary antibodies included ERK1/2 (cell signaling, 4696), pERK1/2 (cell signaling, 9101), PDE8A (protein-tech, 13956–1-AP), c-RAF (cell signaling, 9422), c-RAF (sigma, R2404), c-RAF pS43 (Abcam, ab150365), c-RAF pS259 (cell signaling, 9421), KRAS (protein tech, 12063–1-AP), HSP90 (Santa Cruz, sc-7947).

Techniques: In Vitro, Relative Rate, Western Blot, Staining, Expressing, Derivative Assay, Protein Array

Journal: Scientific Reports

Article Title: Disruption of the pro-oncogenic c-RAF–PDE8A complex represents a differentiated approach to treating KRAS–c-RAF dependent PDAC

doi: 10.1038/s41598-024-59451-3

Figure Lengend Snippet: Schematic illustrating how DRx-170 binds c-RAF, displaces PDE8A and exposes c-RAF to surrounding cAMP microenvironment in the context of KRAS MT cancer. De-protection negatively regulates c-RAF activity in a PKA-dependent manner (pS43/pS259 validated, pS233/pS621 untested), promoting c-RAF conformational closure and dissociation from upstream KRAS. This conservative model depicting DRx-170 mechanism of action highlights how DRx-170 attenuates tumourigenesis through facilitating the allosteric inhibition c-RAF. RBD ras binding domain; CRD cysteine rich domain; AC adenylate cyclase; cAMP cyclic adenosine monophosphate; ATP adenosine triphosphate; AMP adenosine monophosphate; PKA protein kinase A.

Article Snippet: Primary antibodies included ERK1/2 (cell signaling, 4696), pERK1/2 (cell signaling, 9101), PDE8A (protein-tech, 13956–1-AP), c-RAF (cell signaling, 9422), c-RAF (sigma, R2404), c-RAF pS43 (Abcam, ab150365), c-RAF pS259 (cell signaling, 9421), KRAS (protein tech, 12063–1-AP), HSP90 (Santa Cruz, sc-7947).

Techniques: Activity Assay, Inhibition, Binding Assay