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. 2008 Dec 12;417(Pt 1):161–172. doi: 10.1042/BJ20081227

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© 2009 The Author(s) The author(s) has paid for this article to be freely available under the terms of the Creative Commons Attribution Non-Commercial Licence (http://creativecommons.org/licenses/by-nc/2.5/) which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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(A) Intact cell anti-FLAG ELISA screening of the effect of several drugs known to have affinity for the α_1b-adrenoceptor. ELISA assays were performed on cells stably expressing FLAG-α_1b-adrenoceptor TMI-TMIV-eYFP after an overnight treatment with 10⁻⁵ M of each drug. Basal (−) staining reflects non-specific labelling of the cells. Statistics were performed using a one-way ANOVA with the application of the Tukey post-test analysis: *significantly greater than basal, +, significantly lower than the effect of prazosin, P<0.01 in each case. (B) Prazosin increases cell surface FLAG-α_1b-adrenoceptor TMI-TMIV-eYFP in a concentration-dependent manner. Intact cell, anti-FLAG ELISA assays were performed as in (A) following overnight treatment with varying concentrations of prazosin. Results are expressed as mean of the fold increase of the absorbance detected in untreated cells (±S.E.M.). (C) Anti-FLAG immunocytochemistry (α-FLAG, red) was performed as in Figure 1 in fixed, non-permeabilized cells expressing either FLAG-α_1b-adrenoceptor-eYFP (upper images) or FLAG-α_1b-adrenoceptor TMI-TMIV-eYFP (lower images) that were treated overnight with 10⁻⁷ M prazosin. Imaging of eYFP (green) confirmed expression of both constructs and merging of the images (Merge) confirmed that prazosin treatment promoted cell surface delivery of FLAG-α_1b-adrenoceptor TMI-TMIV-eYFP (lower panels, right-hand image). Blue staining represents nuclear staining with the DNA-binding dye Hoechst 33342.