Figure Legends for Supplementary Figures
Suppl. FIG. 1. Internalization of HELor Socby JAWS II cells. JAWS II dendritic cells were incubated with HEL (A) or Soc (B). In each case, cells were incubated with media(a, b) or with25 µg/ml antigen (HEL or Soc;c, d)in a total volume of 0.2 ml for 60 minutes at 37C. Cells were then washed, centrifuged onto coverslips, fixed, permeabilized and treated as described in Materials and Methods. Cells were examined with an Olympus BH2 fluorescence microscope using a 40X objective. JAWS II cells incubated with media alone showed no background staining (panels a). JAWS II cells incubated in the presence of HELor Socshowed bright staining (panels c) indicating that HELand Socwereinternalized. The corresponding bright field images are shown in panels b and d, respectively.
Suppl. FIG. 2. Effect of p24 and Soc on antigen presentation and immunoproteasome subunits.
The experimental set up for antigen presentation is shown in Fig. 3A. The ability of JAWS II cells to present OVA as measured by IL-2 production is shown in panel A. JAWS II cells incubated with E. coli expressed p24 followed by OVA (solid bars) showed a significant decrease in IL-2 production at 12 h and 18 h compared to OVA alone (no p24, white bars, p<0.01). There was no significant decrease in the antigen presentation capacity of JAWS II cells in the presence of Soc (p>0.05). Each bar represents the mean + S.D. of triplicate measurements and one representative experiment out of 2 is shown. *represents significant values compared to the control. In panel B,the effect of p24 produced from various expression systems on the immunoproteasome subunits (2i and PA28) from JAWS II cells is shown. JAWS II cells were incubated with p24 (25 µg/ml) produced in baculovirus, E. coli, yeast, and CHO cells for 18 h. Cell lysates were subjected to 2-dimensional gel electrophoresis followed by Western blotting. The blots were probed with a mixture of anti-mouse 6, 2i, and PA28. The dried blotswere then scanned and analyzed using ImageJ software (NIH, Bethesda, MD) to determine the density of the spots. The density of the spots is expressed as Arbitrary Units. For each culture condition, the 6 subunit of the proteasome in the respective blot was used as the control reference spot and the 2i and PA28 subunits were normalized to the corresponding 6 subunit by dividing the density of each of the specific subunits by the density of the corresponding 6 subunit. One representative experiment out of two is shown. In panel C, the effect of E. coli expressed Soc on the immunoproteasome subunits from JAWS II cells is shown for the following time points: 12 h, 18 h, and 24 h. Cell lysates were subjected to 2-dimensional gel electrophoresis followed by Western blotting as described in Fig. 5. One representative experiment out of two is shown.
Suppl. FIG. 3.Analysis of the immunoproteasome subunits from primary dendritic cells and JAWS II cells following incubation with HIV-1 p24. Cells were incubated with HIV-1 p24 for 90 min in serum-free media, washed and then put back in culture for 12 h. Cells cultured in media without p24 for the entire period served as the control. Cell lysates were subjected to 2-dimensional gel electrophoresis followed by Western blotting. The blots were probed with a mixture of anti-mouse 6, 1i, 2i, 5i, PA28, and PA28. The dried blots were then scanned. In both cell types 2i and PA28 subunits were either considerably reduced or absent. One representative experiment out of two is shown.
Suppl. FIG. 4. Internalization of soluble and liposome-encapsulated p24 in JAWS II cells. JAWS II cells were cultured for 90 min in media alone (shaded area), soluble p24 (grey line), or L(p24 + LA) (black line). Cells were then washed, fixed, permeabilized, stained for intracellular p24, and analyzed on a flow cytometer as described in Materials and Methods.
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