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. 2010 Oct 7;6(10):e1001130. doi: 10.1371/journal.ppat.1001130

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Eyre et al.

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 author and source are properly credited.

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(A) Schematic diagrams of PDZK1, SR-BI and epitope-tagged wildtype and mutant derivatives of each. For SR-BI schematic diagrams transmembrane regions that flank the large extracellular loop are depicted as white boxes. (B) Domains involved in SR-BI/PDZK1 interaction. 293T cells were co-transfected with the indicated SR-BI and PDZK1 expression constructs prior to immunoprecipitation with anti-FLAG antibody and Western analysis of immunoprecipitates using anti-FLAG or anti-Myc antibodies as indicated (lower panels). Note in immunoprecipitate samples PDZ1-FLAG (∼18 kDa) was not distinguishable from light chain bands (not shown). Expression of each of the FLAG- and Myc-tagged proteins, relative to the loading control β-actin, in whole cell lysates (WCL) prior to IP is shown in the upper panel. (C) Detection of serine phosphorylation of PDZK1. 293T cells were transfected with the indicated PDZK1 expression construct prior to immunoprecipitation (IP) with anti-FLAG antibody and Western analysis using anti-FLAG or anti-phosphoserine (SerP) antibodies as indicated. (D) Laser-scanning confocal microscopy (LSCM) analysis of colocalization of overexpressed PDZK1 constructs and full-length SR-BI. Huh-7 cells were grown on gelatin-coated coverslips, transfected with the indicated expression constructs and processed for indirect immunofluorescent detection of FLAG-tagged PDZK1 proteins (green) and SR-BI (red). Merged images (right panels) revealed colocalization (yellow). (E) LSCM analysis of the localization of surface endogenous SR-BI (green) and overexpressed wildtype PDZK1-FLAG (red). Merged images (right panel) revealed colocalization (yellow). For all conditions parallel samples were labelled with secondary antibody alone (surface SR-BI labelling) or isotype-matched irrelevant control antibodies to confirm specificity of labelling (not shown).