FIGURE 3.

In vitro deglycylation assay. A, 20 μl of FLAG affinity-purified gDIP1 and gDIP2 or control immunopurification from the WBC6 strain were incubated as indicated on the top of the panel with 0.2 μg of difopein affinity-purified g14-3-3 from FLAG-gTTLL3-transfected trophozoite in the presence or not of EDTA and separated on 12% SDS-PAGE, transferred on nitrocellulose, and subjected to Western blot with mouse α-polyGly Ab (middle panel) and then with rabbit-g14-3-3 serum (lower panel) and α-HA Ab (upper panel). The brackets (right side) indicate the molecular size range of the polyglycylated g14-3-3; incubation with WBC6 IP (gray bracket) and with FLAG-DIP1 immunoprecipitate (dotted bracket) with or without EDTA and incubation with FLAG-DIP2 immunoprecipitate (black bracket) and with FLAG-DIP2 with EDTA (empty bracket). Molecular size markers are on the left. B, densitometric analysis of the Western blots with α-polyGly and α-g14-3-3 presented in panel A was performed with ImageJ software. In the graphs the peaks represent the optic density (OD) of the immunostained bands reported as arbitrary value (z axes) and the position of the peaks (in cm) correspond to the position of the bands on the membrane (x and y axis). The molecular size shift of polyglycylated g14-3-3 as a consequence of the incubation with FLAG-gDIPs in the presence or absence of EDTA is more evident.