Fig. S1. High-level expression of calsyntenin-1 and KLC1wt induces their colocalisation into abnormal ER structures. Confocal images of CV-1 cells co-transfected with high levels (as judged by brightness of fluorescent signal) of EGFP−calsyntenin-1 and FLAG−KLC1wt and co-stained for the ER marker PDI. Structures unlabelled by PDI probably represent EGFP−calsyntenin-1 and FLAG−KLC1 in Golgi as they are known to also localise to this compartment (Ludwig et al., 2009). Scale bar: 20 m.

Fig. S2. siRNA-mediated knockdown of KLC1 induces perinuclear clustering of calsyntenin-1. HEK-293 cells were co-transfected with EGFP−calsyntenin-1 and either control siRNA (A) or KLC1 siRNA (B) and the distribution of calsyntenin-1 monitored using the EGFP tag. The plasma membranes (red dashes) of representative cells are outlined on phase contrast (left panels) and corresponding EGFP−calsyntenin-1 images (right panels). Treatment with KLC1 siRNA noticeably reduces labelling of calsyntenin-1 in peripheral regions of the cell. Scale bars: 20 m.

Fig. S3. Mutation of KLC1ser460 does not influence KLC1 colocalization with HAP1A in transfected CV-1 cells. (A,B) Confocal images of cells transfected with HAP1A alone (A) or with either KLC1wt, KLC1ser460ala (KLC1ala) and KLC1ser460asp (KLC1asp) (B). Scale bars: 20 m. (C) ICQ values for colocalisation of HAP1A with KLC1wt, KLC1ser460ala and KLC1ser460asp. HAP1A with KLC1wt: ICQ 0.192±0.04, n=10; HAP1A with KLC1ser460ala: ICQ 0.178±0.05, n=10; HAP1A with KLC1ser460asp: ICQ 0.226±0.04, n=10. Values are means±s.d. Data were analysed by one-way ANOVA with LSD post-hoc test (ns, not significant). Error bars are s.e.m.