The IncE sequence used in this study is from the L3 serovar L3/404/LN (NCBI reference WP_015506602) (Harris et al., 2012). The pGEX-4T-2 bacterial expression plasmid encoding the human SNX5 PX domain (residues 22–170) was generated using a standard PCR-based cloning strategy, and its identity confirmed by sequencing. All other bacterial expression constructs for human SNX proteins were synthesized and cloned into pGEX-4T-2 by Genscript (USA). These included the SNX5 PX domain IncE fusion (SNX5 residues 22–170 with IncE residues 108–132 fused at the C-terminus (Figure 4—figure supplement 1A), SNX6 (residues 29–170), SNX32 (residues 17–166), and SNX5 PX domain mutants. The pcDNA3.1-N-eGFP mammalian expression constructs encoding full-length human SNX5, SNX5(F136A), IncE(91-132) and IncE(91-132)(F116D) with N-terminal GFP-tags were generated by Genscript (USA). The pCMU-myc-SNX5 was as described previously (Kerr et al., 2006), and the SNX6 and SNX32 genes cloned into the pcDNA3.1-nMyc vector at BamHI and XhoI restriction sites (Kerr et al., 2012). SNX5, SNX32 and SNX8 were also cloned by polymerase chain reaction, restriction digest and ligation into pEGFP-C1 for expression with N-terminal GFP tags as described previously (Wang et al., 2010).