Figure 1.

Experimental Setup for Protein Turnover Rate Studies in Mice Intestinal Epithelial Cells and Mucus

(A) Conventionally raised (CR) and germ-free (GF) mice were kept for 1 week on an amino-acid-defined diet (Lys [L]). Thereafter, the food was replaced by the same diet where lysine was substituted with ¹³C-lysine (Lys6 [H]). Animals were sacrificed at time points 0, 1, 2, 3, 4, 5, 7, 10, 14, and 32 days after the start of the heavy-labeled diet. Small intestine was divided into five parts and colon into two; caecum was excluded. For the proteomics analysis we used first (duodenum [DE]), third (middle jejunum [MJE]), and fifth (ileum [IE]) part of small intestine and both proximal (PCE) and distal colon (DCE) epithelial cells. Mucus was collected from the ileum and distal colon (IM and DCM, respectively). Mass-spectrometry-based proteomic analyses were used to follow the ¹³C-lysine incorporation into the freshly synthesized proteins, as indicated by the tryptic peptide SGDFELIK from the Muc2 mucin.

(B) Heavy label incorporation into proteins in epithelial cells and mucus. Data points represent the average value of heavy label in at each time point for the different segments and mucus.

See also Figures S1 and S2.