Fig. 4.

SNX27-PDZ domain cargo binding is regulated by Ser⁴⁹ phosphorylation. (A) Caco-2/bbe-SNX27KD cell extracts (1 mg) were incubated with GST or GST fused to PDZ+PX (P-PX, amino acids 1–266), or PDZ+PX-S49A or -S49D (P-PX-A and P-PX-D) (all 1 nmol). The presence of ATP7A, ZO-2 and Glut-1 in the GST pull downs were detected by western blotting (IB, top; representative results from six independent experiments). A quantification of cargo proteins in pull downs normalized to the amount of GST from the 6 experiments is shown below as means±s.e.m. *P<0.05, **P<0.01; ***P<0.001; NS, not significant (compared to WT control). (B) HeLa cells expressing control shRNA, SNX27 shRNA and SNX27 shRNA transfected with SNX27-S49A or -S49D mutant were stained for endogenous GLUT1 (Alexa Fluor 488) and endogenous LAMP1 (Alexa Fluor 594). A representative experiment is shown above with magnified views of regions in the dashed boxes shown. Scale bars: 10 μm. Quantification of colocalization was performed across four independent experiments and is shown below. Results are shown as means±s.e.m. *P<0.05 compared to shRNA control.