Fig. 3.

Identification of an endocytic NP motif in the C-terminal tail of zDHHC2. (A) Amino acid sequence of the 350–366 region of zDHHC2; the NP motif is underlined. (B) Representative confocal images of PC12 cells co-expressing wt mCHERRY-zDHHC2 (left panel) and various GFP-tagged mutants (middle panel). Both are represented in FIRE pseudocolour. The right panel is the merge of the left and middle panels, with the wt mCHERRY-zDHHC2 presented in Magenta and GFP-tagged proteins in Green. Scale bar represents 5 μm. (C) Quantification of the endosomal depletion of the mutants. The fluorescence intensity values of the recycling endosome compartment (endo) as a fraction of the whole cell fluorescence were calculated as described in material and methods for both GFP and mCHERRY. The graph shows the normalized mean ratio ± SEM of endosomal fluorescence of the GFP construct relative to the wt zDHHC2 mCHERRY tagged protein (n = 20 wt zDHHC2 cells, 11 N356A_P358A cells, 11 N357A cells and 10 P358A cells). Statistical analysis (ANOVA) shows a significant difference in the localisation of the mutants compared to wt zDHHC2 (^⁎⁎, p ≤ 0.01; ^⁎⁎⁎, p ≤ 0.001). The dashed and filled lines represent the upper and lower ratios of endosomal enrichment that were calculated in Fig. 1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)