Skip to main content
. Author manuscript; available in PMC: 2012 Jan 1.
Published in final edited form as: J Immunol. 2010 Nov 22;186(1):403–410. doi: 10.4049/jimmunol.1001906

Figure 1. PMN-contact induces short form of IRAK-M and CASP-6 activation in alveolar macrophages.

Figure 1

(A) Western blot probed with IRAK-M antibody. Alveolar macrophages from a volunteer without lung disease (lane 1) were cultured with PMN at a ratio of 1/1 for 4 h. After incubation, protein extract from macrophages was subjected to western blot with anti-IRAK-M antibody. BAL cells of 5 separate patients with pneumonia were processed immediately post bronchoscopy (lanes 3-7). The same membrane was re-probed with anti-β-actin as a loading control. Densitometry reveled that relative actin expression in these clinical samples with differing red blood cell contamination of pneumonia patient BAL cells was 1.9, 1.9, 1.0, 1.9 and 1.2. (B) Western blot probed with IRAK-M antibody. Alveolar macrophages from two additional volunteers without lung disease (lane 1) were cultured with differing PMN ratios for 4 h or stimulated with three concentrations of LPS, in ng/ml. The same membrane was re-probed with anti-β-actin as a loading control. (C) Western blot probed with IRAK-M antibody. Alveolar macrophages from two additional volunteers without lung disease (lane 1) are shown. A 0.4 micrometer filter was placed above the AM (lane 2). PMN at a 5/1 ratio for 4 h were added directly to the AM (lane 3) or placed in the filter insert above the AM (lane 4). The same membrane was re-probed with anti-β-actin as a loading control. (D) Western blot probed with IRAK-M antibody. Cell lysate was prepared from unstimulated THP-1 macrophages, the protein extract was separated in the sucrose gradient centrifuge and fractionated from the top of tubes (fractions 1-5 represent membrane bound protein and fractions 10-12 represent soluble unbound proteins). The same membrane was re-probed with anti-Lyn, a protein that binds to detergent resistant membranes. The Lyn signal is a control for the quality detergent resistant membrane separation by the sucrose density gradient. Representative data of three independent experiments are shown. (E) Western blot probed with IRAK-M antibody. THP-1 macrophages 1h after addition of isolated PMN membrane. Extracts were separated on a sucrose gradient as described in panel D. The Lyn re-probe of the membrane is shown as a loading control.