Overexpression of catalytically active PATs increase the amount of palmitoylated D2L.

<p>(A-C) myc-tagged PATs or a catalytically inactive variant of GODZ, GODZ (DHHS), were transiently expressed in HEK-D2L cells. Untransfected cells were used as a control. (A) Protein expression of D2R in lysates was analyzed via SDS-PAGE and Western blotting (top). Normalized amounts of 15-HDYA-labeled D2Ls were IP’d, and fluorescently imaged (middle). Total D2L in IPs was analyzed by Western blotting (bottom). (B) Normalized amounts of 15-HDYA-labeled D2Ls were IP’d, fluorescently imaged (top) and total D2L in IPs was analyzed by Western blotting (bottom). (C) Expression of D2R (top) and GODZ or GODZ (DHHS) (bottom) in lysates was analyzed via SDS-PAGE and Western blotting. Quantitation of receptor expression was normalized to GAPDH expression for protein (top graph) and mRNA (bottom graph). D2R mRNA expression levels in cells overexpressing GODZ or GODZ (DHHS) were determined by RT-qPCR and normalized to untransfected levels. (A-C) Brackets indicate bands corresponding to D2R that were used for protein quantitation. Levels of palmitoylated D2L were normalized to the amount of IP’d receptor (A & B). Bar graphs represent the average pixel density (as determined by ImageJ) from three experiments. Data was analyzed using a two-sided unpaired Student’s <i>t</i> test (expressed as ± SEM, <i>n</i> = 3, *P < 0.05).