WT MSD MPS-IIIA. Lysosomal ph
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1 7 6 Lysosomal ph Supplementary figure S1. Lysosomal ph was measure in, and MPSIIIA MEFs using a Lysosensor yellow/blue-dextran (Molecular Probes) as a lysosomal ph indicator (see methods). P <.5, Student s t-test: vs and vs.
2 β-gal β-hex β-glu ENRICHMEN T HGSNAT ARSC Supplementary Table T1. To determinate the purity of lysosomal fraction we measured the enrichment and the recovery of specific lysosomal and non lysosomal enzymes in each fraction obtained after the magneticpurification (Postnuclear surnatant, ; Flow-through, ; Lysosomal lumen, ; Lysosomal membrane, ). Lysosomal Standard fluorometric assays were performed to determine the activities of the following enzymes: β-hexosaminidase (β-hex) and β-galactosidase (β-gal) (which are both present in the lysosomal lumen); β-glucosidase (β-glu) and heparan acetyl CoA: α-glucosaminide N-acetyltransferase (HGSNAT) (which are localized to the lysosomal membrane); and arylsulfatase C (ARSC) (which is found in the membrane of the endoplasmic reticulum). The column represents that calculated to be present in each fraction, describes the fold increase in arbitrary activity in the sample as a percentage of that in the and RECOVERY refers to the amount of enzymatic activity in a given fraction relative to that in the represented as a percentage. These results are representative of 3 separate experiments in MEFs. Very simal results were obtained after lysosomal magnetic purification from LSD MEFs.
3 ,5 1 3, cholesterol EGF (hr) EGFR actin % of EGFR (vs T) T,5h 1h 3h time of EGF incubation + cholesterol,5 1 3,5 1 3 EGF (hr) + MβCD EGFR actin % of EGFR (vs T) T,5h 1h 3h time of EGF incubation + MβCD Supplementary figure S2. EGFR degradation was followed in and MEFs after treating the cells with cholesterol (A) or MβCD (B) respectively. The cells were loaded with EGF for the indicated time to stimulate EGFR internalization. The cells were then immediately lysed and subjected to anti-egfr blot. The amount of remaining EGFR was quantified by densitometry analysis (Image J) of the blot and expressed in the chart as % of the EGFR amount present at time T (1%). The values in the chart represent the mean ± S.E.M. of three independent experiments. P <.5, Student s t-test: vs + cholesterol and vs + MβCD at each time point.
4 VAMP7 GM1 Supplementary figure S3. VAMP7 accumulates in the DRMs in LSD cells.vamp7 co-localization with DRMs was visualized in / MEFs and in MEFs by co-immunofluorescence analysis between VAMP7 and GM1 raft lipid (labeled with a red-fluorescent cholera toxin B). Scale bar, 1 µm.
5 BOILED NON BOILED kda SDS-resistant complexed form monomeric form 25 lysosomal membrane WB: anti-syntaxin 5 Supplementary figure S4. SDS-resistant complexes containing syntaxin 5 were detected by immunoblotting analysis of non-boiled total membrane samples derived from and MEFs
6 zoom golgin-97 LAMP1 Vti1b/golgin-97 Vti1b/LAMP1 Vti1b golgin-97 LAMP1 Vti1b/golgin-97 Vti1b/LAMP1 Vti1b golgin-97 LAMP1 Vti1b/golgin-97 Vti1b/LAMP % of Vti1b co-localizing with LAMP1 % of Vti1b co-localizing with golgin-97 Vti1b Supplementary figure S5., and MPSIIIA MEFs were subjected to a triple labeling with anti-vti1b, anti-golgin-97 (a marker of TGN) and LAMP1 antibodies. The double merges between Vti1b and either golgin-97 or LAMP1 were quantified in 15 different cells and displayed as % of Vti1b co-localizing with either golgin-97 or LAMP1(means ± SEM ). See also enlarged images showing the severe reduction of Vti1b/golgin-97 co-localization in LSD MEFs. P <.5, Student s t-test: vs and vs. Scale bar, 1 µm.
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Boucher et al NCOMMS B
1 Supplementary Figure 1 (linked to Figure 1). mvegfr1 constitutively internalizes in endothelial cells. (a) Immunoblot of mflt1 from undifferentiated mouse embryonic stem (ES) cells with indicated genotypes;
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DOI:.38/ncb2822 a MTC02 FAO cells EEA1 b +/+ MEFs /DAPI -/- MEFs /DAPI -/- MEFs //DAPI c HEK 293 cells WCE N M C P AKT TBC1D7 Lamin A/C EEA1 VDAC d HeLa cells WCE N M C P AKT Lamin A/C EEA1 VDAC Figure
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SUPPLEENTRY INFORTION DOI: 1.138/ncb2577 Early Telophase Late Telophase B icrotubules within the ICB (percent of total cells in telophase) D G ultinucleate cells (% total) 8 6 4 2 2 15 1 5 T without gaps
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