Volume 8 issue 12 December 2007
A Heparan Sulfate-Facilitated and Raft-Dependent Macropinocytosis of Eosinophil Cationic Protein
Tan-Chi Fan, Hao-Teng Chang, I-Wen Chen, Hsiu-Yiu Wang and Margaret Dah-Tsyr Chang

Supplemental Figure 1. Flow cytometry analysis of (A, B) 100 µg/ml Tfn-AF 555 (blue line/without acid wash, red line/with acid wash) and (C, D) 0.1 µg/ml CtxB-AF 555 (blue line/without acid wash, red line/with acid wash) uptake for indicated time points in Beas-2B cells after preincubation for 30 min in the presence of CPZ (cyan line), NYS (purple line), or MbCD (green line).  The black line corresponds to cells incubated in the absence of Tfn or CtxB. Results are expressed as mean fluorescence normalized to untreated control. A typical plot of cell number versus mean fluorescence is shown in B and D (20 min internalization).

Figure 1 (.jpg)

Supplemental Figure 2. ECP uptake in Eps15- and dynamin-independent.  CHO-K1 cells transfected with a GFP-tagged control (DIIId2) or a GFP-tagged dominant-negative (EH29) Eps15 construct were seeded on coverslips. After 24 h, the cells were incubated with MBP-ECP (Cyan) and Tfn-AF 555 (Tfn; red) for 15 min at 37°C, acid stripped, fixed and processed for MBP-ECP detection by confocal microscopy. Transfected cells were identified by GFP expression (arrows). CHO-K1 cells were transfected with a Dyn-HA or a K44A Dyn-HA dominant-negative construct. After 36 h, the cells were incubated with MBP-ECP (cyan) and Tfn-AF 555 (red) for 15 min at 37°C, acid stripped and fixed. Transfected cells were identified by HA expression (arrowheads). Cells were incubated with rabbit anti-MBP and mouse anti-HA antibodies followed by staining with donkey anti-rabbit-Cy5 and goat anti-mouse-FITC secondary antibodies, respectively. Scale bar, 10 µm.

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Supplemental Figure 3. The distribution of MBP-ECP (cyan) together with Tfn-AF 555 (red, A) or CtxB- AF 555 (red, B) was examined after 10 min incubation in Beas-2B cells transfected with CHC-siRNA-2 (A), Cav-1-siRNA-2 (B), or sc-siRNA as a control. CHC and Cav-1 were detected by immunofluorescence using mouse anti-CHC and rabbit anti-Cav-1 antibodies followed by staining with goat anti-mouse-FITC and goat anti-rabbit-FITC IgG, respectively. Scale bar, 10 mm.

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Supplemental Figure 4. A, flow cytometry analysis of 1 mg/ml FITC-Dextran, 70 kDa (red line) uptake for 15 min in Beas-2B cells or after preincubated for 30 min in the presence of nocodazole (green line), cytochalasin D (purple line), amiloride (blue lines), or LY294002 (cyan line). The black line corresponds to cells incubated in the absence of FITC-Dextran. B, histograms indicate mean and deviations (s.d.) of two independent experiments for each drug treatment, shown as percent of mean fluorescence normalized to untreated control.

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