Mast cells were cultured from mouse bone marrow in 50% WEHI-3B-conditioned medium as described by Razin et al. (1984). (A) RT-PCR for cathepsin L and cathepsin S produced amplimers of predicted size on total RNA harvested from wild-type (WT) but not cathepsin L/S^-/- BMMCs. Total RNA was isolated from cathepsin L/S^+/+ or cathepsin L/S^-/- BMMCs using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA), and then reverse-transcribed into single-stranded cDNA using Superscript II reverse transcriptase (BD Biosciences, Palo Alto, CA, USA) and oligo dT primers. Cathepsin L and S transcripts in this cDNA were detected by PCR using the following primers: cathepsin L: forward, 5′-GTT GTG TGA CTC CTG TGA AG-3′; reverse, 5′-CTT GCT GCT ACA GTT GGG T-3′; cathepsin S: forward, 5′-CCA AGT GGG CAT GAA CG-3′; reverse, 5′-GCC AGT AAT CTT TGC CAT CAA G-3′. (B) Active site [¹²⁵I]-JPM labeling of cysteine proteases. Cathepsin L/S^+/+ or cathepsin L/S^-/- BMMCs (2×10⁶ /sample) were labeled by culturing cells in the presence of [¹²⁵I]-JPM ethyl ester for 24 h at 37°C as described by Liu et al. (2006). Cells were washed twice with PBS, lysed in SDS-PAGE sample buffer, and labeled proteins were analyzed by 12% SDS-PAGE and autoradiography.

Sections of mouse stomach (A,C) and ear (B,D) obtained from cathepsin L/S^-/- (A,B) or DPPI^-/- (C,D) mice. Harvested tissues were fixed for 6–18 h in PBS containing 4% paraformaldehyde. Tissues were embedded directly in Tissue-Tek OCT compound (Miles, Elkhart, IN, USA), frozen at -80°C, and then sectioned (5 μm). Prior to use, these sections were equilibrated in PBS. To detect active chymase in tissue sections, chloroacetate esterase histochemistry was performed as described by Leder (1979). Slides were then washed with water, counterstained in 0.5% eosin Y, mounted, and photographed. Note the esterase-positive mast cells (arrowheads) in tissue sections obtained from cathepsin L/S^-/- (A,B) but not DPPI^-/- mice (C,D). Images were obtained using a 20× objective lens and are representative of observations made in six animals.

Samples of 10 μg of total protein from cell lysates obtained from cathepsin L/S^+/+, cathepsin L^-/-, cathepsin S^-/- or cathepsin L/S^-/- BMMCs and purified dog DPPI were subjected to SDS-PAGE under reducing conditions and transferred to polyvinylidine difluoride membranes (Life Sciences Products, Boston, MA, USA). The membrane was washed with 50 mm Tris-HCl containing 0.5 m NaCl, 0.01% Tween-20 (TBS; pH 7.5), and incubated for 1 h in TBS containing either a 1:1000 dilution of rabbit anti-bovine DPPI (a gift from John Hoidal, University of Utah, Salt Lake City, USA) or monoclonal anti-β-actin antibody (Sigma-Aldrich). The membrane was then washed with TBS, incubated in TBS for 30 min containing a 1:2000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG (New England Biolabs, Beverly, MA, USA) and washed again. Immunoreactivity was detected using the phototope-HRP detection kit (New England Biolabs). Note that bands of immunoreactive DPPI appear at similar sizes in both cathepsin L/S^+/+ and cathepsin L/S^-/- BMMC lysates.