Supporting Information

Files in this Data Supplement:

Fig. 6. Diagram of BPAG1n4 protein structure. Amino acid residue numbers denote boundaries for the functional domains. Id, first exon of BPAG1n4; ABD, actin-binding domain; MTBD, microtubule-binding domain.

Fig. 7. Vesicular localization of retrolinkin in axons at ultrastructural level. (A-F) Immuno-EM analysis of retrolinkin's subcellular localization in sciatic nerves. Retrolinkin labeling with anti-RTLN was visualized with 15-nm gold particles conjugated with secondary antibody. (G-I) Double immuno-EM labeling of mouse sciatic nerve sections with anti-RTLN (15 nm) and anti-EEA1 (5 nm). Open arrows indicate colocalization of retrolinkin and EEA1. (J) Negative control, secondary antibodies alone. [Bar: 100 nm (A-E), 50 nm (F), 130 nm (G), 65 nm (H-I), and 200 nm (J).]

Fig. 8. Retrolinkin localizes to vesicular structures. (A-C) Double immunofluorescence staining of EEA1 (early endosome antigen) (A) and flag-tagged retrolinkin (B) in transfected COS7 cells. (D-F) Shown are double staining of Golgi apparatus (D) and flag-retrolinkin (E). Inset in C shows high magnification of merged image. (Bar: 9 mm.)

Fig. 9. Retrolinkin costains with BPAG1n4 in cultured DRG neurons. (A-C) Colocalization of retrolinkin and BPAG1n4 in cultured DRG neurons. (D-F) Double immunostaining of NF-L and retrolinkin. (G-I) Double immunostaining of Bip and retrolinkin. Boxed inset shows colocalized retrolinkin and BPAG1n4. [Bar: 20 mm (A-I).]

Fig. 10. Neurons overexpressing retrolinkin-cyto-GFP are viable. Shown are Trypan blue (Invitrogen) staining of DRG neurons expressing GFP (A and B) or retrolinkin-cyto-GFP (C and D), and dead neurons with high autofluorescence (E and F). Cells were stained with 0.2% Trypan blue for 10 min at 37°C, washed with PBS, and fixed with 4% paraformaldehyde.