File S1 - Uptake and Intracellular Trafficking of Superantigens in Dendritic Cells

Contains: Figure S1. Supplementary figure to Figure 1 A and B. SAg incorporation in DCs. (A) DC CD11c-PE isotype control; (B) CD11c expression and SEG incorporation in DC cultured at 37°C without SEG-FITC, and (C) with 50 µg/ml of SEG-FITC. Percentage of DC incorporating 50 µg/ml SEG-FITC in the presence of (D) WT, (E) EIPA or (F) at 4°C. Figure S2. Supplementary figures to Figure 1D, E and F. SAg incorporation in DCs and cell activation markers. Bone marrow DCs were pulsed 1 h with SEG-FITC, washed, and immunolabeled with anti CD11c-FITC. (D) Cell surface MHC-II molecules on DCs treated with SSA, SEG, SEI or LPS for 24 h, compared to non-treated basal control, and expressed as percentage of positive cells. (E) CD80 on SAgs- or LPS-treated DCs compared to non-treated. (F) Endocytosis of OVA-FITC by SAg or LPS pre-treated DCs. (I) CD86 on SAgs- or LPS-treated DCs compared to non-treated. (J) CD40 on SAgs- or LPS-treated DCs compared to non-treated. Figures show a representative experiment of 3–5. *p<0.05, **p<0.01. Figure S3. DCs re-expose SEG on plasma membrane. DCs were pulsed with SEG for 1 h, cultured further for the indicated times (0–240 min), and incubated with anti-SEG polyclonal antibodies and specific FITC conjugated antibodies without fixation and permeabilization treatment. The presence of SEG at the DC plasma membrane after 3 h confirms that the SAg was re-exposed on the cell membrane.
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