Fig. 9. SNX5 forms heteroligomers with SNX1. (A) Full-length SNX1 and SNX5 were fused to either the LexA DNA-binding domain in the pLexA vector or the B42 activation domain in the pYESTrp vector as indicated. Interactions were assessed using a liquid galactosidase assay and reported in Miller units (MU). Values are mean ± s.e.m. (B) Total extracts of HEK-GFP-SNX5 cell monolayers were prepared as described in the Materials and Methods, and lysates immunoprecipitated with an anti-GFP polyclonal antibody. Immune complexes were collected and subjected to SDS-PAGE under reducing conditions. After transfer to PVDF membranes, membranes were immunoblotted with monoclonal antibodies raised against SNX1 and HRP-conjugated anti-mouse immunoglobulin using a chemiluminescence detection system. Molecular mass in kDa is indicated on the left. (C) Total extracts of HEK-GFP-SNX5 cells treated with scrambled or SNX1-specific siRNA duplexes for 96 hours were prepared as described in the Materials and Methods and separated by SDS-PAGE. The relative amount of endogenous SNX1 and SNX2 in the samples was then determined by western immunoblotting with monoclonal anti-SNX1 and SNX2 antibodies. (D) HEK-GFP-SNX5 cell monolayers treated with scrambled or SNX1-specific siRNA duplexes for 96 hours were fixed in 0.9% PFA and labelled with a mouse anti-SNX1 monoclonal antibody followed by Cy3-conjugated goat anti-mouse IgG. Bar, 10 µm.