Fig. 1

Lack of interaction of αSyn and VPS35 in human cells. A-B) Co-IP assays between V5-tagged human VPS35 (WT, D620N and P316S) and (A) untagged or (B) Myc-tagged human WT αSyn indicate a lack of interaction of VPS35 with αSyn. HEK-293T cell extracts co-expressing V5-tagged VPS35 (WT, D620N or P316S) and WT αSyn were subjected to IP with anti-V5 antibody, and IP and input fractions were probed for αSyn, Myc (αSyn), VPS26 and V5. VPS35 variants interact equivalently with endogenous VPS26 but not with αSyn. C-D) Overexpression of PD-linked αSyn variants does not influence the steady-state levels of endogenous retromer subunits. C-D) Western blot analysis of Triton-soluble extracts from SH-SY5Y cells transiently expressing untagged human αSyn variants (WT, A53T, A30P or E46K) or empty vector. Graphs indicate the levels of each retromer subunit, VPS35, VPS26 or VPS29, normalized to β-tubulin levels (mean ± SEM, n = 3 experiments). Data were analyzed by one-way ANOVA with Dunnett’s post-hoc analysis. ns, non-significant. E) Knockdown of endogenous VPS35 does not increase the levels of endogenous or overexpressed human WT αSyn. Western blot analysis of Triton-soluble extracts from SH-SY5Y cell clones stably expressing miR30-adapted shRNAs targeting VPS35 (#1 or #2) or a non-silencing control shRNA. VPS35 levels are markedly reduced in cells expressing VPS35-shRNAs together with corresponding reductions in VPS26 and VPS29, as expected, whereas overexpressed αSyn is also reduced. F) Western blot analysis of SH-SY5Y cells stably expressing VPS35-shRNAs reveal a similar reduction of overexpressed Myc-tagged human WT tau