Figure 2
From: Somatic cell hemoglobin modulates nitrogen oxide metabolism in the human airway epithelium
Heme proteins in human airway epithelial cell cultures were detected by immunoblot, immunofluorescence and by LC–MS proteomics. (A) Hbβ was immunoprecipitated (IP Hbβ) and run with total lysate from cells (TLC) and whole erythrocyte lysate (RBC) on an SDS gel, followed by Coomassie blue staining. Three bands were excised from the immunoprecipitated Hbβ (IP Hbβ) band and TLC for LC–MS. LC–MS analysis of the samples reveals: Band A: Hbβ, Hbδ, Hbα, Protein S100-A6. Bands B and D: Hbβ, Hbδ, Hbα and other proteins. (B) Airway cell proteins have bound heme. Drabkin reagent (Sigma, # D5941) was used to determine average amount of heme/total protein15. Three normal primary human airway epithelial cell samples, consisting of 10–12 filter each scraped in RIPA buffer, were lyophilized and reconstituted in Drabkin reagent. Heme was measured with absorbance (λ = 540 nm) by spectrophotometer. The fibroblast NIH 3T3 cells were used as a control. (C) Immunofluorescent staining of primary normal human airway epithelial cells grown for 35 days at ALI, then fixed, permeabilized and labeled as described in the methods, confirmed expression Hbβ in HBE cells (top). (Nucleus—DAPI, blue; cilia—white, HBβ-green, f-actin red). (D) Isotype control IgG (for the anti-Hb Ab) in cells grown identically to those in 2D served as a negative control.
