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Fig. 2. PX domain-dependent direct interaction between SNX16 and PtdIns(3)P. (A) The ability of GST-fused SNX16^WT and SNX16^Y145A to bind a variety of phosphoinositides (PI) was analyzed by protein-lipid overlay assay. 100 pmols of the relevant PI was spotted on to a nitrocellulose membrane, which was then incubated with the purified proteins. The membranes were washed and proteins bound to the membrane by lipid interaction were detected with anti-GST antibody. Results representative of at least three separate experiments are shown. (B) COS-7 cells were transfected with GFP-tagged SNX16 and fixed. Cells were processed to detect SNX16 (green) and TfR (red) simultaneously by confocal microscopy. Like TfR, SNX16 was redistributed from endosomal membranes to the cytosol after treatment with wortmannin. Bars, 10 µm. (C) COS-7 cells were transfected with FLAG-tagged SNX16^WT and SNX16^Y145A, cultured in the absence of serum for 3 hours and then treated with or without wortmannin (100 nM) for 15 min. Cells were lysed and lysates were immunoreacted with anti-TfR antibody. Complexes associated with TfR were analyzed with anti-FLAG and anti-TfR antibodies. Total cell lysates (40 µg) were immunoblotted with anti-FLAG antibody.

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