Fig. 4: Excess aS decreases the interaction between SNX1 and VPS35 and inhibits the retrograde transport.

A Confocal microscopic images of Dox-inducible aS-expressing SH-SY5Y cells triple-immunostained for M6PR (green), TGN46 (magenta), and aS (cyan). Upon aS induction, M6PR showed a more dispersed distribution near the cell periphery compared to the uninduced cells. The white dotted line indicates the cell periphery. Scale bar: 20 μm. B The colocalization rate of M6PR and TGN46 was slightly, but significantly, reduced by aS induction. Data were statistically analyzed using the Mann–Whitney U test. *p < 0.05, n = 51 (non-induced) and 51 (induced). C Co-IP analysis of binding between aS and SNX1 in the cytoplasmic fraction of the aS-induced cells. The arrow and arrowhead indicate the bands of SNX1 and IgG heavy chain, respectively. D Results of the WB analysis of the aS-induced and non-induced cells solubilized with saponin and Triton X- 100. The early endosomal marker Rab5 was detected in the Triton- but not the saponin-soluble fraction, indicating that the former mainly contains cytoplasmic proteins and the latter membrane-associated components. Upon aS induction, SNX1 in the saponin-soluble cytosolic fraction increased, while SNX1 in the triton-soluble membrane fraction remained unchanged. β-tubulin is an indicator of equal loading. E The ratio of SNX1/β-tubulin band intensity. Values of Dox-induced cells are expressed relative to the values of Dox-uninduced cells which are presented as 1.0, and the calculated values are expressed as the mean ± standard error. Data were statistically analyzed using the Student’s t test. *p < 0.05, n = 4. F Interaction between VPS35 and SNX1 before and after aS induction and verified by co-IP. The amount of SNX1 bound to VPS35 was slightly decreased after the aS induction.