Figure 3. The interaction of SNX27 with FAM21 is required for proper recycling of its cargo to the plasma membrane (PM).

(a) Schematic representation of SNX27 constructs used. (b) hTERT-RPE1 cells stably expressing empty vector or 3 × FLAG-tagged, siRNA-resistant SNX27 constructs were transfected with indicated SNX27 or control (siCTL) siRNAs, and analysed by immunoblotting. An arrow denotes endogenous SNX27. The SNX27 ΔPDZ proteins were not detected by anti-SNX27 antibodies because of a deficiency of epitope. (c) Myc-FAM21 was co-transfected with the indicated HA-SNX27 constructs in HEK293T cells, and lysates were immunoprecipitated with control mouse IgG or anti-haemagglutinin (HA) antibodies. Then, precipitates were immunoblotted as indicated. Asterisks (*) denote IgG heavy or light chains. (d) hTERT-RPE1 cells stably expressing empty vector or 3 × FLAG-tagged, siRNA-resistant SNX27 constructs were transfected with the indicated siRNAs and immunostained for FLAG epitope (green) and GLUT1 (red). Merged images with 4',6-diamidino-2-phenylindole staining (blue) are to the right. Scale bar, 10 μm. (e) Cells were counted based on co-localization of GLUT1 with GM130 (Golgi) or LAMP1 (lysosome) as shown in Supplementary Fig. 3. Cells without accumulated GLUT1 signals were considered as plasma membrane (PM). Graphs express means±s.d. (n=3; 200 cells per group). (f) hTERT-RPE1 cells stably expressing empty vector or 3 × FLAG-tagged, siRNA-resistant SNX27 constructs were transfected with the indicated siRNAs. Twenty-four hours later, cells were cultured in the absence or presence of 50 nM bafilomycin A1 for 24 h, and then analysed by immunoblotting. The intensities of each GLUT1 were normalized with respective GAPDH signals. Levels of GLUT1 in siSNX27-transfected cells relative to siCTL-transfected cells were measured from five independent experiments. Graphs express means±s.d. *P<0.05, **P<0.01; NS, not significant.