SNX17 is required specifically in the ligand-expressing cells for Notch signaling. (A) Knockdown of SNX17 by siRNAs in 293 T cells down-regulated the Notch luciferase reporter activity while over-expression of SNX17 stimulated the reporter activity. (B) Over-expression of Jag and Delta family Notch ligands stimulated the reporter activity which was blocked by the knockdown of SNX17. (C) Down-regulation of SNX17 reduced the expression of endogenous Notch target genes hes1/7 and hey1 in 293 T cells as determined by real-time RT-PCR (p < 0.05 in all cases). hey2 was not affected in the same assay (P > 0.1). β-actin was the reference. (D) Co-culture of the Jag1-expressing 293 T cells with the Notch1-expressing NIH3T3 cells. Knockdown of SNX17 in the ligand-expressing cells reduced the Notch reporter activity while down-regulation of SNX17 in the receptor-expressing cells did not have inhibitory effect. All assays were repeated at least three times and data represent mean ± SD from three independent experiments.