-and NaF evoke LPA-mimicking [35 S]GTPγS binding response that is sensitive to LPA
receptor antagonist. (a) Horizontal brain sections were incubated using a three-step autoradiography protocol as described in Methods. AlFx- (NaF 10 mM + AlCl3 50 μM) was included during the 40 min preincubation step (step 1), NaF (10 mM) was included in step 2, and LPA (50 μM) in step 3, whereas Ki16425 (5 μM) was present throughout all steps. The buffer in all the steps additionally contained 0.1% BSA. Treatment with AlFx- and NaF results in region-specific G protein activity that is restricted to the LPA1 receptor enriched white matter tracts, a response that is mimicked by exogenous LPA and is sensitive to Ki16425 (cc, corpus callosum; fi, fimbria of the hippocampus). Scale bar = 5 mm. (b) and (c) Quantitative data of the binding responses. Autoradiography films were digitized and [35 S]GTPγS binding was quantified from corpus callosum of the coronal sections of 4 week-old rat brain, as detailed in Methods. Data are calculated as nCi/g equivalents with non-specific binding subtracted from the total and are expressed as a percentage of basal binding (mean + SEM) from six individual animals (n = 6). Significance level: (b) ***p < 0.001 compared to control in each LPA concentration (c) ***p < 0.001 compared to the treatment without Ki16425.