Mouse MA10 Leydig tumor cells were a kind gift of Dr. Mario Ascoli (University of Iowa). Rolipram was purchased from Tocris Bioscience (Bristol, UK). PF-04957325 was a kind gift of Pfizer Inc. (Groton, CT). Isotope labeled amino acids for SILAC were obtained from Cambridge Isotope Labs (Andover, MA) or Sigma-Isotec (St Louis, MO). Custom RPMI medium (−Lys/−Arg) was obtained from Caisson Labs (North Logan, UT). Dialyzed FBS and PHOS-Select Iron affinity gel were from Sigma (St. Louis, MO). C18 reverse phase material for nano-LC columns (3 µm Reprosil-C18.aq) was obtained from Dr. Maisch (Ammerbuch, DE). Protease and phosphatase inhibitor cocktails as well as the Pierce 660 nm protein assay were purchased from Thermo Scientific (Rockford, IL). Sequencing grade endoproteinase LysC and Trypsin were obtained from Promega (Madison, WI). Oasis C18 cartridges for peptide extraction were from Waters (Milford, MA).
Pf 04957325
Manufactured by Pfizer
PF-04957325 is a laboratory product manufactured by Pfizer. It is a piece of lab equipment designed to facilitate scientific research and analysis. The core function of this product is to assist researchers in their work, but a detailed description of its intended use cannot be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Rolipram, by Bio-Techne (1 mentions)
Dialyzed fbs, by Merck Group (1 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Pierce 660 nm protein assay, by Thermo Fisher Scientific (1 mentions)
Phos select iron affinity gel, by Merck Group (1 mentions)
Gonal f, by Merck Group (1 mentions)
11 prohu e01, by ALPCO (1 mentions)
Raf 1, by Cell Signaling Technology (1 mentions)
Goat anti mouse igg hrp, by Thermo Fisher Scientific (1 mentions)
P44 42 mapk, by Cell Signaling Technology (1 mentions)
Phospho p44 42 mapk thr202 tyr204, by Cell Signaling Technology (1 mentions)
Anti cd3 antibody, by BioLegend (1 mentions)
4 protocols using pf 04957325
1
Proteomic Analysis of Mouse MA10 Leydig Tumor Cells
2
Porcine COC Progesterone Secretion Assay
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As previously described, a commercial immunoassay (ALPCO Diagnostics, Salem, NH, product number 11-PROHU-E01) was used27 (link) to measure the progesterone secreted by porcine COCs cultured in North Carolina State University-23 (NCSU-23) medium47 (link) without bovine serum albumin containing 25 µM of 2-mercaptoethanol, 0.1 mg/ml of cysteine, 10% (v/v) filtered porcine follicular fluid, and 0.01 µg/ml of recombinant human FSH (GONAL-f, Serono, Mississauga, ON, Canada)47 (link). This was done in the presence or absence of the PDE8 inhibitor (PF-04957325, donated by Pfizer under a Material Transfer Agreement). The concentration of PF-04957325 (300 nM) corresponded to the cell based IC9023 (link). For each experimental condition, 10 COCs were incubated for 48 h in an equal volume (125 µL) of medium in 96-well assay plates. Maturation medium and COCs were recovered from three independent experiments and kept at −80 °C until use. The progesterone in the maturation medium was assayed in triplicate according to the manufacturer’s instructions. The sensitivity of the assay was 0.1 ng/mL.
3
PDE8 Inhibitor PF-04957325 Protocol
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The PDE8-specific inhibitor, PF-04957325, was donated by Pfizer under a Material Transfer Agreement.
4
Characterization of VCAM-1, ICAM-1, and PDE8A-Raf-1 Interactions
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Recombinant mouse VCAM-1/CD106 Fc chimera (643-VM), recombinant mouse ICAM-1/CD54 Fc chimera (796-IC) were purchased from R&D systems, Minneapolis, MN. Protein A was purchased from Sigma-Aldrich (P7837). The primary antibodies for phospho-p44/42 MAPK (Thr202/Tyr204) (9101; 1:1000), p44/42 MAPK (9102; 1:1000), Phospho-Raf-1 (pSer259) (9421; 1:1000) and Raf-1 (9422; 1:1000) were purchased from Cell Signaling Technology. An additional Raf-1 antibody was purchased from BD Biosciences (610152; 1:2000). Anti-GAPDH (ab75834; 1:100,000) and anti-PDE8A antibodies (1:1000) were purchased from Abcam and Scottish Biomedical, respectively. The anti-CD3 antibody was purchased from Biolegend (100331). The secondary antibodies goat anti-rabbit IgG-HRP (65-6120, 1:4000), chicken anti-rabbit IgG-HRP (sc-2963, 1:5000) and goat anti-mouse IgG-HRP (sc-2005, 1:5000) were purchased from Invitrogen and Santa Cruz Biotechnology. The PDE8A-Raf-1 disruptor peptide (PDE8A: R454-T465; RRLSGNEYVLST) and scrambled control peptide (SYTVRLLGERNS) were synthesized as described [19] . The PDE8 selective inhibitor PF-04957325 was synthesized by Pfizer Inc., Groton, CT [6] .
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