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. 2006 Apr 17;203(4):973–984. doi: 10.1084/jem.20050625

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Copyright © 2006, The Rockefeller University Press

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Figure 8. — Postnatal depletion of IRAK-1 in IECs. (A) Immunoblot for IRAK-1 in primary IECs isolated from fetal (day −1), 1-, 3-, and 6-d-old mice. A secondary antibody control was included to demonstrate the specificity of the IRAK-1 staining. Actin was included as loading control. (B) Analysis of IRAK-1 expression in LMD-isolated primary IECs. HPRT was included as housekeeping control. (C) Immunostaining for IRAK-1 in small intestinal tissue of fetal (day −1), 1-, and 6-d-old mice. A peptide control illustrates the specificity of the immunstaining. Counterstaining with DAPI. Bar, 100 μm. (D–F) Immunoblot for IRAK-1 in macrophage-like RAW 264.7 cells and mouse intestinal epithelial m-IC_cl2 cells at the indicated time points (hours) after exposure to 100 ng/ml LPS. (G) Immunoblot for IRAK-1 in mouse intestinal epithelial m-IC_cl2 cells 2 h after exposure to 100 ng/ml LPS in the absence or presence of 25 μM of the kinase inhibitor K-252b. Actin was included as loading control. (H) Immunoblot for IRAK-1 in mouse intestinal epithelial m-IC_cl2 cells left untreated or stimulated with 100 ng/ml LPS for 6 h (left). m-IC_cl2 cells transfected with an ubiquitin expression plasmid and stimulated with 100 ng/ml LPS for 0 or 0.5 h were immunoprecipitated using a polyclonal anti-ubiquitin antibody and immunoblotted to visualize IRAK-1 (right). Note the size difference between native and ubiquitinated IRAK-1. HC, heavy chain; LC, light chain.