Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cathepsin S controls MHC class II-mediated antigen presentation by epithelial cells in vivo.

doi: 10.4049/jimmunol.174.3.1205

Figure Lengend Snippet: FIGURE 1. Ii processing is im- paired in Cat S/ IEC. Sections from B6, Cat S/, and Cat L/ mice of small intestine (A) and large intestine (B) and sections from B63B6, B63Cat S/, Cat S/3B6, and Cat S/3Cat S/ chimeric mice of small intestine (C) and large intes- tine (D) were stained with I-Ab (Y3P) and mouse CLIP:I-Ab (15G4) com- plex-specific mAbs.

Article Snippet: The Y3P mAb recognizes I-Ab (ATCC HB183), MKD6 recognizes I-Ad (ATCC HB3), and the YAe mAb recognizes I-Ab with E peptide (aa 52–68) bound in the peptide groove (17).

Techniques: Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cathepsin S controls MHC class II-mediated antigen presentation by epithelial cells in vivo.

doi: 10.4049/jimmunol.174.3.1205

Figure Lengend Snippet: FIGURE 2. Analysis of Cat S and Cat L levels and Ii degradation intermediates in IEC from B6 and Cat S/ mice. A, IEC (2 105) were incubated for 2 h with the irreversible cysteine protease inhibitor Cbz-[125I]-Tyr-Ala-CN2. Cells were lysed, and radiolabeled enzymes were analyzed on a 12% SDS-PAGE gel. Cat B served as an internal control, because comparable levels of activity were present in IEC from both types of mice. The positions of Cat B and Cat S in the gel are indicated by arrows. B, The level of Cat L in B6 and Cat S/ IEC was detected by immunoblotting with a Cat L-specific polyclonal antiserum. Cells (2 105) were solubilized in lysis buffer, and the proteins were separated by PAGE. The arrow indicates mature Cat L. These experiments were performed three times and yielded identical results. C, Lysates of 2 105 IEC from B6 and Cat S/ mice were separated by SDS-PAGE and immunoblotted with the mAb IN-1. The positions of Ii fragments p41, p31, and p12 are indicated. D, IEC from six to eight B6 and Cat S/ mice were analyzed by flow cytometry, and the level of expression of MHC class II (Y3P) and CLIP I-Ab (15G4) on the surface of IEC was determined by gating on CD45negG8.8pos cells. The data shown are representative of five independent experiments. E, Ultrathin cryosections of B6 and Cat S/ IEC were immunolabeled with M5/114 and IN-1 using 10-nm protein A gold particles. Partial cell profiles are shown. Labeling for M5/114 and IN-1 was observed in dense lysosomal multivesicular compartments. The micrographs are representative of 100 cell profiles examined for each mouse phenotype.

Article Snippet: The Y3P mAb recognizes I-Ab (ATCC HB183), MKD6 recognizes I-Ad (ATCC HB3), and the YAe mAb recognizes I-Ab with E peptide (aa 52–68) bound in the peptide groove (17).

Techniques: Incubation, Protease Inhibitor, SDS Page, Control, Activity Assay, Western Blot, Lysis, Cytometry, Expressing, Immunolabeling, Labeling