*2.7. Protein Kinase A (PKA) Activity*

Epimastigotes cells (5 × 107 cells/mL) were washed twice in ice-cold phosphate buffer saline (PBS, pH 7.2) and lysed in 0.5 mL radioimmunoprecipitation assay buffer (RIPA buffer) for 30 min. PKA activity was assayed in the presence of 4 mM Hepes-Tris (pH 7.0), 0.4 mM MgCl2, 1 mM CaCl2, 1 μM ATP, and 50 μg of lysed cells in a final volume of 50 μL, in the presence or absence of 5 μM 3-isobutyl-1-methylxanthine (IBMX, a PKA activator), in MTS-11C mini tubes (Axygen Scientific, Union City, CA, USA). The reaction was triggered by adding 50 μL of the Kinase-Glo luminescent kit, and after 10 min at 37 ◦C, the samples were placed in a GloMax Multi JR detection system (Promega Corporation, Fitchburg, WI, USA). PKA activity was quantified as the difference between the reading in the presence of IBMX and in the absence of the activator.
