FIGURE 3.

The SNX27-β-Pix interaction is direct and specific for the β-Pix isoform containing the carboxyl-terminal PDZ binding motif. a, schematic diagram of two variants of β-Pix. Variant 1 (β1-Pix) is shorter and contains a C-terminal type I PDZ binding motif sequence (ETNL). Variant 3 (β2-Pix) is longer and lacks this binding motif (SHQN). b, 1 μg of cDNAs of β-Pix variant 1, variant 3, or a mutant variant 1 lacking the C-terminal PDZ binding motif (ΔETNL) were translated in the presence of [³⁵S]methionine. The translated product was incubated with GST or GST fusion protein encoding the PDZ domain of SNX27 for 1 h at 4 °C. The beads were washed, and bound translated product was separated by SDS-PAGE. Detection of bound product was revealed by autoradiography. Twenty percent of the translated product was used as input control. c, mpkCCD cells were transiently transfected with empty HA-tagged vector or plasmids containing HA fusion proteins of the indicated β-Pix sequences. After 48 h incubation, the mpkCCD cell lysates were incubated with GST or a GST-SNX27 PDZ. Expressed fusion protein bound to GST/GST-PDZ was identified by Western blotting using an HA-specific antibody (top). Equal GST loading was confirmed by Coomassie staining (bottom). 20 μg of protein lysate was used as input.