Journal: Computational and Structural Biotechnology Journal

Article Title: Plasticity and therapeutic potential of cAMP and cGMP-specific phosphodiesterases in Toxoplasma gondii

doi: 10.1016/j.csbj.2022.09.022

Figure Lengend Snippet: Catalytic activity of the native phosphodiesterases present in tachyzoites of T. gondii . (A) Scheme for the α-HA agarose bead-based immunoprecipitation to enrich the smHA-tagged PDE proteins from the cell lysate of transgenic parasites. The parental strain ( RH Δ ku80 Δ hxgprt ) was used as a negative control. (B-D) Immunoblots confirming adequate precipitation of PDE-smHA proteins. Samples from panel A were probed with the mouse α-HA (green) and rabbit α- Tg Hsp90 (red) antibodies. Note the presence of Tg Hsp90 (a cytosolic marker) in cell-free extract (CFE) and its absence in the immunoprecipitated pellet (P). Asterisks, if shown, mark the predicted size of smHA-tagged PDEs. (E) Phosphodiesterase activity of PDE-smHA proteins with cAMP and cGMP. The colorimetric enzyme assays were set up using 6 μg of PDE samples ( Tg PDE5, 10 μg) and 200 μM substrate (1 h, 37 °C). The control reactions run alongside lacked the substrate or enzyme. The substrate-free enzyme-only reaction was subtracted from samples to quantify the PDE activity (normalized to the protein amount). The negative controls indicate the precipitated protein of the parental strain (N.D., not detectable). The data show the mean ± SE (n = 3–4 assays).

Article Snippet: After washing with 0.1 % BSA in PBS (pH 7.4), they were incubated for 30 min with rabbit anti-mouse bridge antibody (BioZol original from Jackson-Immuno, 315–005-048), diluted 1:100 in 1 % BSA, 0.2 % fish skin gelatin in PBS (pH 7.4).

Techniques: Activity Assay, Immunoprecipitation, Transgenic Assay, Negative Control, Western Blot, Marker, Control