Volume 8 issue 8 August 2007
Innate Immune Recognition Triggers Secretion of Lysosomal Enzymes by Macrophages
Rebecca L. Lackman, Amanda M. Jamieson, Hans Geuze, Peter Cresswell

Supplemental Figure 1.  CI-M6PR-reactive GILT is expressed in the B-cell line 45.1.    Approximately 3 million 45.1 cells were extracted in TBS/1% TX-100 and subjected to 4 rounds of sequential pull-down with either MIg-agarose control beads or CI-M6PR beads.  The eluate of the final pulldowns is shown in Lanes 1 and 2.  Residual GILT was immunoprecipitated from the depleted samples with MaP.IP30 beads, and the bead eluate was resolved in Lanes 3 and 4.  All samples were run under reducing conditions and blotted with R. GILT serum.  Blots were developed with alkaline phosphatase coupled goat anti-rabbit antibody and ECF reagent.  Quantification of the signal revealed that approximately 65% of cellular GILT was CI-M6PR-reactive.

Figure 1 (.jpg)

Supplemental Figure 2. Localization of GILT in IFN-γ stimulated cells.
THP-1 cells were stimulated with 200 units/mL IFN-γ for 48 h, seeded onto alcian-blue-coated coverslips, fixed, permeabilized and stained for total GILT (a) and precursor GILT (b) (left panels) and LAMP2 (middle panels), followed by species-specific secondary fluorescent F(ab’)2 fragments.  The merged images are displayed in the right panel.  Both images were acquired at 63x magnification, with an optical zoom of 4.3.  The confocal slice shown is representative of the staining observed in multiple fields.

Figure 2 (.jpg)

Supplemental Figure 3.  Double-labeling of bacterially-stimulated THP-1/GILT for precursor GILT and the CI-M6PR.
THP-1/GILT cells were stimulated with E. coli (MOI 2) for 24 h and processed for immunoelectron microscopy.  Cryosections were immunolabeled for precursor GILT (15 nm gold) and CI-MPR (10 nm gold).  Precursor GILT is present in the Golgi stack (G), but is absent from the CI-MPR positive clathrin-coated TGN vesicles (arrows). Bar, 200 nm.

Figure 3 (.jpg)