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. Author manuscript; available in PMC: 2018 Jun 1.
Published in final edited form as: J Cell Physiol. 2016 Oct 26;232(6):1404–1414. doi: 10.1002/jcp.25634

Figure 5. PKA inhibition by H-89 caused pHi acidification and Em depolarization.

Figure 5

Human sperm were incubated for 1 hour in media that supports capacitation in the absence (CAP) or presence of different concentrations of H89 (10, 30 and 50 μM). Aliquots from each condition were processed for Western blotting with anti-pPKA antibodies and then membranes were reblotted with an anti-β-tubulin antibody for loading control. (A) PKA activity was inhibited at 30 μM of H89. At this concentration, intracellular acidification (B) and depolarization (C) was observed when compared to the control condition (CAP). cAMP agonists (dbcAMP, 1 mM + IBMX 0.2 mM, dotted line) did not rescue the acidification nor the depolarization produced by H89 (30 μM) as expected. Blot and histograms are representative of at least 4 experiments. (D) Relative median compared to the control condition (CAP) of BCECF and DiSC3(5) fluorescence of samples incubated with H89 in the presence or absence of 1 mM dbcAMP and 0.2 mM IBMX.