Cysteine and Aspartyl Proteases Contribute to Protein Digestion in the Gut of Freshwater Planaria
Fig 6
Immunofluorescence labeling and electron microscopy of zymogen and catalytic SmedCB shows strong labeling in cells lining the intestinal lumen.
A) Several antibodies were generated against Schmidtea cathepsin B, including one against the catalytic region only (underlined region), and one against the zymogen tag and catalytic region (a combination of the green and pink peptides). (B) Western blot with antibody used to localize SmedCB at 1:5,000. Lane 1, recombinant prep of SmedCB from E. coli. Band at 70kDa is a dimer of SmedCB, lower band at 35kDa is zymogen SmedCB. Lanes 2–5 contain planaria lysate at 15μL, 7.5 μL, 5μL, and 15μL, respectively. Higher band (~37kDa) is zymogen, lower band (~27kDa) is catalytic domain. Lanes 1–4 use the mouse-derived antibody underlined in A, while lane 5 was blotted against the rabbit-derived antibody from the pink and green peptides in A. (C) Labeling of paraffin-embedded S. mediterranea cross sections of worms starved for one week with (1:100) anti-SmedCB zymogen pro-peptide antibody showed strongest signal in the cells lining the intestinal lumen. Comparison with the negative control (D) anti-rabbit secondary (1:100) only shows that the labeling on the border of the planaria cross sections is due to drying artifact. This pattern is also seen in worms with the anti-SmedCB catalytic domain only antibody (1:100) (E). The negative control (F) with anti-mouse secondary only (1:100), shows labeling only on the edges of the cross sections due to drying artifact. Worms were also starved for two weeks prior to labeling (G) as opposed to one week in C and E. SmedCB labeling remained the same. (H) Cross sections of S. mediterranea worms (starved one week) were labeled with (1:100) anti-SmedCB zymogen pro-peptide antibody. Intestinal lumen seen as white space in the center of the image. (I) Immuno-gold labeling of antibody target, visualized as black dots, shows vesicle-like structures are heavily labeled. Other tissue, like the rounded, gray lipid droplets in (J) did not show labeling.