JCS03316 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Homology of BAD-LAMP with other LAMP family members. (A) Comparison of BAD-LAMP sequences with others members of LAMP family and DC-HIL. All alignments were done using EMBOSS needle (Needleman-Wunsch global alignment) to obtain a percentage of similarity. (B) BAD-LAMP conservation among species. All alignments were done using SSEARCHp able to calculate identity percentage. Drosophila melanogaster peptide ID: CG32225-PA; Takifugu rubripes peptide ID: NEWSINFRUP00000148857; Tetraodon nigroviridis peptide ID: GSTENP00020285001; Ratus norvegicus peptide ID: ENSRNOP00000007274; Anopheles gambiae peptide ID: ENSANGP00000022286; Homo sapiens peptide ID: ENSP00000246070; Mus musculus peptide ID: ENSMUSP00000061180; Gallus gallus: ENSGALP00000014483; Bos taurus: ENSBTAP00000010487

• Supplemental Figure 2 -

  Fig. S2. Summary of molecular markers and BAD-LAMP distribution in cortical neurones. A list of the different molecules imaged by confocal microscopy in cortical neurones is given along with their level of co-localization with BAD-LAMP and a representative staining (BAD-LAMP in red, markers in green).

• Supplemental Figure 3 -

  Fig. S3. Tyrosine 276 is necessary for BAD-LAMP endocytosis in neurones. (A) Internalization of FLAG antibody (red) and cholera toxin (green) in FLAG-BAD-LAMP-transfected neurones for indicated time. Bar, 20 μm. (B) Cortical neurones co-transfected with BAD-GFP and FLAG-BAD-LAMP cytoplasmic tail tyrosine 276 mutant (Tyr-276-Ala) were visualized by confocal microscopy. Staining for FLAG antibody (red), BAD-GFP (green) and LAMP2 (white). Bar, 20 μm. (C) Internalization of FLAG antibody (red) in Tyr-276-Ala-transfected neurones and staining for LAMP2 (white). Tyrosine 276 is necessary for proper BAD-LAMP sorting in neurones and endocytosis. Bars, 20 μm.

• Supplemental Figure 4 -

  Fig. S4. BAD-LAMP recycles in transfected HeLa cells. (A) Subcellular Percoll gradient fractionation showing that in transfected HeLa cells BAD-LAMP is mostly localized in light density fractions and is absent from heavy density fractions containing the lysosomal enzyme β-hexosaminidase. (B) Representative FACS staining experiment (of three), showing the level of internalized Flag antibody against time at the surface of FLAG-BAD-LAMP-transfected HeLa cells. The timing of internalization and the recovery of surface staining at 10 minutes indicates the BAD-LAMP recycles in HeLa cells. (C) Control experiment demonstrating inhibition of transferrin uptake (red) after dynamin A44K expression (green). (D) Control experiment demonstrating inhibition of transferrin uptake (red) by AP2 RNAi transfection (green).