Fig. 5: Phosphorylation affects GD-NT localization and oligomerization.

A Detection of GD-NT in culture medium. HEK293 cells were transfected with the indicated constructs. 16 hours later, the culture medium and WCL were harvested and immunoblotted with indicated antibodies. B Distribution pattern of GD-NT-WT or its mutants. HeLa cells were transfected with the indicated constructs. Shown are representative confocal microscopy images of the distribution of ectopic Flag-GD-FL, GD-NT-WT, GD-NT-S46D or GD-NT-S46E (green) co-stained with DAPI (blue). C Subcellular localization of GD-NT-WT or its mutants. HEK293 cells were transfected with the indicated constructs. 16 hours later, cells were harvested and subjected to sample preparation using Thermo Fisher Subcellular Fractionation Kit. Membrane fractions were immunoblotted with indicated antibodies. WCL was included as an input control. D, Oligomerization of GD-NT. HEK293 cells were transfected with the indicated constructs. 16 hours later, cells were harvested and subjected to IB analysis with indicated antibodies under reducing or non-reducing conditions. E Fractionation via SEC/gel filtration. HEK293 cells were transfected with the indicated constructs. 16 hours later, cells were harvested for fractionation with SEC/gel filtration. Eluent protein fractions were immunoblotted with indicated antibodies. Input represents 10% of proteins used for SEC/gel filtration. Data are representative of at least two independent experiments.