All growth media contained equal concentration of basal salts (2.25 g/L NaCl, 0.105 g/L KCl, 0.12 g/L CaCl2, 0.05 g/L NaHCO3).
Wild-type cells carrying control vector or either GAL1-HMO1, GAL1-NHP10, or GAL1-IXR1 were grown to log phase in selective media containing raffinose and equal numbers of cells were spotted to control media (pre-gal samples).
The latter is in agreement with the study of Yoshimura et al. [ 14] who found a MIC of 0.5 μg/mL for A. pyogenes and in accordance with a report by Narayanan et al. [ 15], that support our finding that bovine A. pyogenes strains are able to grow on the selective media containing 16 μg/mL bacitracin (equals approximately 1 U/mL).
Equal volumes of media containing AP or AP-tagged proteins were pre-cleared with Protein A-Agarose beads at 4°C for 2 h and then incubated with the LRP4ecto-bead conjugates at 4°C overnight.
For LPS stimulation, the splenocytes from 1-month old MRL-lpr mice were adjusted to 5×106 cells/ml, plated in 24-well plate, and then activated for 24 hrs by adding equal volumes of media containing LPS (Sigma, 1000 ng/ml) to seeded cells (final concentration at 500 ng/ml).
The LRP4ecto-Fc medium was incubated with Protein A-Agarose (Sigma) at 4 C for 2 hrs to make LRP4ecto-Fc-Agarose conjugates and equal volumes of media containing AP or AP-tagged proteins were precleared with Protein A-Agarose at 4 C for 2 hrs prior to incubation with the conjugates at 4 C overnight.
To investigate binding of AP-tagged proteins to LRP4 in cell system, HEK293A cells were transfected with pCDNA3-LRP4 fulengthgth constructs for 48h, washed once with PBS plus 0.1% BSA, and incubated in equal volumes of media containing AP or AP-tagged proteins at 37°C for 1 h.
To test binding of AP and AP-tagged proteins in cell system, HEK293A cells were transfected with pCDNA3.1-LRP4 constructs for 48 hrs, washed once with PBS plus 0.1%BSA, and incubated in equal volumes of media containing AP or AP-tagged proteins at 37 C for 1 hr.
The diluted serum samples were mixed with an equal volume of media containing homotypic influenza virus at a concentration of 1050%% tissue culture infectious dose/well.
The DNA was mixed with 2 μl of Lipofectamine 2000 reagent (Life Technologies, Rockville, MD, USA) in a total volume of 50 μl of serum-free RPMI per cm2 of cells, incubated for 3 hours, and followed by the addition of an equal volume of media containing 20% fetal calf serum.
