The Journal of Neuroscience, November 8, 2006, ():
Perturbed Interactions of Mutant Proteolipid Protein/DM20 with Cholesterol and Lipid Rafts in Oligodendroglia: Implications for Dysmyelination in Spastic Paraplegia
J. Neurosci. Krämer-Albers et al.
26: 11743
Supplemental Data
Files in this Data Supplement:
- supplemental material
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Supplemental Material Figure 1: Cell surface versus intracellular localisation of wild-type PLP/DM20 in primary oligodendrocytes and transfected Oli-neu cells. Primary cultured oligodendrocytes (pOL), untransfected Oli-neu cells, PLP-transfected, or DM20-transfected Oli-neu cells were co-stained with antibodies recognising different epitopes of PLP/DM20. Live cells were stained with the O10 antibody, which recognises a cell surface epitope and thus selectively stains PLP/DM20 localised in the plasma membrane (red, cell surface PLP/DM20). After surface staining, cells were fixed and permeabilised and stained with the AA3 antibody, which binds to the cytoplasmic C-terminus of PLP/DM20 thus recognising all intracellular PLP/DM20 molecules in addition (green, total PLP/DM20). Endogenous PLP in primary oligodendrocytes as well as ectopically expressed PLP/DM20 in Oli-neu cells is efficiently transported to the plasma membrane. A considerable fraction of the protein is localised to intracellular membranes.
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Supplemental Material Figure 2: Endosomal/Lysosomal localisation of PLP in a subpopulation of COS7 cells. Wild-type PLP transfected COS7 cells were labelled with the vital dye LysoTracker (B,C, red) and stained with antibodies AA3 against PLP (A,C, green). The arrowhead marks a cell with the typical localisation of PLP in the ER. The arrow points to a cell, where PLP in addition is localised to endosomes/lysosomes. A higher magnification of the boxed area is shown in C. Bar 30 µM in A and 5 µM in C.
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Supplemental Material Figure 3: Equal transfection efficiency and expression levels of wild-type and mutant PLP/DM20 in Oli-neu cells. A, Wild-type, rsh- and msd-PLP-transfected cells (identical transfections to the experiment shown in Fig. 4C) were stained with AA3 antibodies recognising PLP/DM20. The staining was performed after the 3H-photo-cholesterol labelling and images have been acquired with identical exposure times. Wild-type and mutant PLP/DM20 are expressed with equal efficiency (transfection efficiency of 61 +/-2,3%) and the steady state expression levels are comparable. B, Immunoprecipation of PLP/DM20 from wild-type and mutant PLP/DM20 transfected cells followed by western blot with the AA3 antibody detecting immunoprecipitated PLP. The average yield of PLP/DM20 in such an experiment is similar. However, the yield of msd-PLP is slightly reduced. LC, light chain; * marks PLP/DM20 dimer. Bar: 100 µM.
