Fig. 2
From: Modifying Rap1-signalling by targeting Pde6δ is neuroprotective in models of Alzheimer’s disease
REM bind to the prenyl-binding pocket of Pde6δ. a Concentration-dependent interaction of methotrexate fused REM compound (REM0044931) to full-length Pde6δ in the mammalian 3-hybrid assay MASPIT (EC50 = 45 nM; n = 3). b Pde6δ-Strep and REM compound interaction assay. Left, schematic representation: E. coli lysates producing Pde6δ-Strep, E. coli lysates with His-tagged DHFR and 10 μM REM were mixed and subjected to a Strep-Tactin pull-down. Right, co-precipitation of DHFR-His was assessed by Western analysis. Pull-down with all compounds as described above (lane AS), without REM-MTX (lane A) or without Pde6δ-Strep (lane B). c Left, genetic silencing of PDE6δ in the toxicity assay in function of increasing concentrations REM (n = 6). Box on the right, efficiency of silencing validated by Western blot ((representative immunoblots; immunostaining of GAPDH was determined as a loading control; Pde6δ expression was reduced on average by 88% ± 3%; Average ± SEM) (One-way ANOVA (REM treatment); siNC: P = 0.0002; F = 5.29; DF = 7; siPDE6δ: n.s.)). d Superimposition of REM compound (purple) and farnesyl (grey). e REM docking into the Pde6δ prenyl-binding pocket showing all interacting amino acids. In green indicates hydrophobic amino acids, in dark blue positively charged amino acids and in cyan neutral polar amino acids. f REM docked in Pde6δ’s prenyl-binding pocket (ribbon)