FIGURE 3.

Inhibition of PDE activity and activation of adenylyl cyclases increase PKA activity levels, disrupt cell polarization, and alter the gradients of PKA activity. Clone A cells were treated with 1 mm IBMX (A) or 1 mm IBMX plus 25 μm forskolin (B) and plated on laminin-1-coated coverslips in the presence of the same concentrations of drugs and imaged as described in Fig. 1. Line graphics below the Fc/YFP images represent the average pixel intensity of the Fc/YFP ratio versus the distance in microns. Bar graphics represent fluorescence intensity for YFP and Fc/YFP for regions noted by the red boxes as displayed in Fig. 1. The bar in the left-hand Fc/YFP panel of A indicates 10 μm and is representative of the scale for all images. C, Clone A cells treated with 1 mm IBMX or 1 mm IBMX plus 25 μm forskolin or left untreated were plated on laminin-1-coated dishes for 30 min then extracted, and active PKA levels relative to total PKA were measured using a pseudosubstrate affinity assay. Glutathione S-transferase-PKI bound PKA (Active) and lysate controls (Total, representing 10% input) were immunoblotted for the PKA catalytic subunit and then quantified (graphs). Note that immunoblots are from the same gel, same exposure. Representative data are shown.