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. Author manuscript; available in PMC: 2017 Oct 1.
Published in final edited form as: Neurobiol Aging. 2016 May 21;46:22–31. doi: 10.1016/j.neurobiolaging.2016.05.016

Fig. 5.

Fig. 5

The D620N mutation in VPS35 impairs its regulation on DRD1 trafficking and downstream signaling. (A) VPS35(D620N)-myc and DRD1-HA vectors were co-transfected into HEK293T cells for 24 h. Cell lysates were subjected to immunoprecipitation (IP) with anti-HA antibody, anti-myc antibody or mouse IgG (mIgG). VPS35(D620N) and DRD1 in immunoprecipitated proteins were immunoblotted with anti-myc and anti-HA antibodies, respectively. (B) N2a cells were transfected with control, wild-type (WT) VPS35-myc and VPS35(D620N) vectors for 24 h, and then subjected to surface biotinylation. Affinity precipitated biotinylated proteins, as well as proteins in total lysates were analyzed by Western blot. (C,D) HEK293T cells were transfected with DRD1-HA vector and split equally. After second transfection with VPS35(D620N)-myc and control vectors for 24 h, cells were equally split again and biotinylated for surface proteins at 4°C. Biotinylated cells were incubated at 37°C for indicated time periods and then treated with glutathione at 4°C to cleave biotin on remaining surface proteins (C), or incubated at 37°C for 30 min, treated with glutathione at 4°C to cleave biotin on remaining surface proteins, incubated for indicated time periods, and then treated with glutathione at 4°C again to cleave biotin on proteins recycling back to cell surface (D). After lysing and affinity precipitation, biotinylated DRD1-HA was analyzed by Western blot with anti-HA antibody. (E) N2a cells were transfected with control, WT VPS35-myc and VPS35(D620N) vectors for 24 h. Cell lysates were assayed for indicated proteins by Western blot. (F) N2a cells were transfected with VPS35 shRNA1 (sh1) and mock shRNA vectors for 60 h. After equal splitting, cells were transfected with control, WT VPS35-myc and VPS35(D620N) vectors for another 24 h. Cell lysates were assayed for indicated proteins by Western blot. Protein levels were quantified by densitometry for comparison to those of controls (set as one arbitrary units), n=3, *p<0.05, **p<0.01; n.s.: not significant.