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Figure 2 | Veterinary Research

Figure 2

From: Differential response of bovine mammary epithelial cells to Staphylococcus aureus or Escherichia coli agonists of the innate immune system

Figure 2

Detection of MAMP in SaS and effect on MEC. a) Detection of LTA in SaS. SaS (50 μL) was submitted to SDS-PAGE and immunoblotted with a mAb to LTA (track 1). LTA appeared as a smear owing to its size heterogeneity as a result of varied number of glycerolphosphate units. Immunoblotting was also performed with SaS treated with proteinase K (track 2) or with purified S. aureus LTA (250 ng or 150 ng, tracks 3 and 4). SaS was depleted of LTA with beads coated with mAb to LTA (track 5). High molecular weight bands are the mAb light and heavy chains. SaS treated with control beads without mAb is shown (track 6). b) MEC were incubated for 16 h with SaS (25%) treated with control beads or with mAb-coated beads, and concentrations of CXCL8 in cell culture supernatants measured by ELISA. Purified LTA (250 ng/mL) was added to the depleted SaS to restore activity. c) Staphylococcal protein A (SpA) was detected in SaS and was able to stimulate bMEC. Immunoblot of 8-h N305 SaS revealed with a peroxidase-conjugated rabbit antiserum reacting with SpA; d) bMEC were incubated for 6 h with either recombinant SpA or purified SpA, and CXCL8 concentrations measured in the cell culture supernatant by ELISA. e) Dose–response of bMEC to decreasing concentrations of staphylococcal alpha hemolysin. Bovine MEC were incubated with purified alpha hemolysin for 16 h, and CXCL8 was measured in cell culture supernatant by ELISA. To investigate the heat-resistance of the stimulus, alpha hemolysin was heated at 95°C for 10 min.

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