Figure 5. Syn5 interacts with GPP130.

(a) Western blotting of a representative GFP-Syn5 pull-down experiment in HeLa cells. GFP-Syn5 cells were treated with 0.05% DMSO (control), 25 μM Retro-2, or siRNAs against Syn5 or GPP130. Note that pull-down of GPP130 was only observed in the control condition. For Syn5, the double bands represent the two isoforms of the protein. Blot is representative of 4 independent repeats. See Supplementary Fig. 7c for full gels. (b) Quantification of GPP130 signals on western blots following GFP-Syn5 pull-down in the indicated conditions. Graph shows mean +/- SD each point represents one experiment, from 4 independent experiments. One-way ANOVA was performed overall (P < 0.0001) with Dunnett’s multiple comparisons test comparing GFP-Syn5 to Retro-2 or Retro-2.1 treated. *** P < 0.0001. (c) Schematic representation of Syn5 and GPP130 constructs purified for in vitro binding studies. (d) Purified Syn5_202-355 was incubated with purified GST, GST-GPP130_1-108 (WT), GST-GPP130_1-108 with KR_11,12AA substitution in the cytosolic domain, or GST-GPP130_1-108 with a substituted cytosolic domain from DPPIV (DGG). Anti-GST-beads were used to collect the complexes, recovery of Syn5_202-355 was determined by Coomassie staining of SDS-PAGE gels, and quantified from 6 independent experiments. Graphs show mean +/- SD, each point represents one experiment. Conditions normalized to total loaded protein (Load). One-way ANOVA was performed overall (P < 0.0002) with Sidak’s multiple comparisons test. NS P = 0.9925, *** P < 0.001, ** P = 0.0012. See Supplementary Fig. 7d for full gels. (e) Experiment as in (d) in which the pull-down of Syn5_202-355 by GST-GPP130_1-108 (GPP130-WT) was quantified in the presence of 25 μM Retro-2 or 0.05% DMSO. Graphs show mean +/- SEM, DMSO and Retro-2 conditions normalized to total loaded protein (Load). Paired two-tailed t-test comparing DMSO to Retro-2, NS P = 0.5. See Supplementary Fig. 7e for full gels.