Prenatal cocaine exposure did not alter the expression but markedly increases the synaptic membrane distribution of GRASP-1.

<p>(<b>a</b>) Expression levels of GRASP-1 in the post-mitochondrial synaptosome-enriched fraction prepared from frontal cortices (FCX) of P21 prenatal saline- and cocaine-exposed rats were compared by Western blotting. Blots were stripped and re-probed with anti-β-actin to validate equal loading. Blots are representative for each treatment group. (<b>b</b>) Densitometric quantification of GRASP-1 expression levels in FCX of P21 prenatal cocaine- vs. saline-exposed rats showed no significant differences. <i>n</i> = 4. Data are means ± s.e.m. of the ratios of the indicated protein to loading control. (<b>c</b>) Membranous (M) and cytosolic (Cy) distribution of GRASP-1 in the FCX of P21 prenatal cocaine- and saline-exposed rats. Solubilized proteins (50 µg) were size-fractionated on 7.5% SDS-PAGE and Western blots were performed with antibody against GRASP-1. Blots were stripped and sequentially re-probed with anti- GRIP1 (2618±177 and 2405±159 optical intensity in saline and cocaine, respectively) and -Caspase3 (1159±131 and 1210±118 optical intensity in saline and cocaine, respectively) antibodies to validate equal loading in M and Cy extracts. Blots are representative for each treatment group. (<b>d</b>) Densitometric quantification of GRASP-1 cellular distribution blots. <i>n</i> = 4. Data are means ± s.e.m. of the optical intensity. *p<0.01 compared the level of GRASP-1 in cytosolic and membrane fractions in the FCX of P21 prenatal cocaine- vs. saline-exposed rats. (<b>e</b>). Immunohistochemical detection of GRASP-1 confirms a higher membrane-localized GRASP-1 in the FCX of P21 prenatal cocaine- vs. saline-exposed rats. The representative sections show more GRASP-1 puncta in membrane of cocaine-treated rats. Scale bar = 50 µm. <i>n</i> = 4 for each group.