Employing a equivalent assay, the capacity of IBH derived PrPSc to drive the amplification of PrPres has been shown to reduce in the absence of NaCl

ell culture supernatants applying a bead assay (MILLIPLEX MAP MMP Magnetic Bead Panels, EMD Millipore, Billerica, MA) with the Bio-Rad Bio-Plex 200 method (Bio-Rad, Hercules, CA) or with commercially out there ELISAs (CXCL5 and SCF; R&D Systems). BALF gelatinase activity was determined by gelatin zymography [13]. Densitometry was performed on MMP9 positive bands and represented densitometry units (DU) measured by pixel intensity of bands, applying Bio-Rad Laboratories Image Lab software (version 4.0, build 16). BALF cells were analyzed for apoptotic cells utilizing the LIVE/DEAD cell viability assay from Invitrogen on the Guava easyCyte flow cytometer from Millipore. The apoptotic cells were expressed as a percentage of total BALF cells. Monolayers of human small airway epithelial (SAE) cells from healthy subjects directly obtained from Lonza (Walkersville, MD, catalogue # CC-2547) were submerged cultured as previously described [22, 23]. All cells are assayed and test negative for HIV-1, mycoplasma, Hepatitis-B, Hepatitis-C, bacteria, yeast and fungi upon isolation by Lonza. Cells were only used for experiments at Eupatilin passages 3 and at a confluency of approximately 70%. SAE cells were transfected by administering silencing RNA (siRNA) specific for p38, MMP9 or negative control scrambled (Scr.) (Qiagen, Gaithersburg, MD). Cells were infected with RSV at a MOI of 0.3 or treated with mock or human MMP9 protein for 24 hours. Human MMP9 (Abcam; ab168863) was activated by incubating with 2mM 4- aminophenylmercuric acetate (APMA; Sigma) at a 9:1 (MMP9:APMA) ratio for 90 minutes at 37 and inactivated with 2mM EDTA. Active and inactive MMP9 were dialyzed twice in TBS to remove APMA or EDTA prior to treating cells or RSV. RSV was treated with various concentrations of active and inactive MMP9 prior to infecting SAE cells. TCID50 assays were performed to determine the quantity of virus following MMP9 treatment. TCID50 data was calculated utilizing the method of Reed and Munch [24]. SAE cells were also treated with active and inactive MMP9 alone and TCID50 assays were performed. Lung tissue protein from mice was homogenized in radio-immunoprecipitation assay (RIPA) buffer, centrifuged at 13,000 x g for 10 minutes and supernatants collected. Lung protein extracts were assayed for myeloperoxidase (MPO) activity using a kit from Cayman Chemical Company (Ann Arbor, MI) as recommended by the manufactures. p-p38 and p38 levels in mouse lung lysates and BMDM were examined using a beads assay together with the Bio-Plex 200 method (Bio-Rad). Activity was represented as fluorescent intensity of the phosphorylated proteins over total proteins. Immunoblots were also conducted to determine levels of p-p38 (Thr180/ Tyr182) (Catalogue #4511; monoclonal antibody produced from rabbits immunized with synthetic phosphopeptide corresponding to residues surrounding Thr180/Tyr182 of human p38 MAPK; final concentration 1/1000 dilution), p38 MAPK (Catalogue #9212; polyclonal antibody produced from rabbits immunized with synthetic peptide corresponding to the sequence of human p38 MAPK; final concentration 1/1000 dilution), MMP9 (Catalogue #2270; polyclonal antibody produced from rabbits immunized with synthetic peptide corresponding to amino acid residue Gly657 of human MMP9; final concentration 1/1000 dilution) and -actin (Catalogue #4967; polyclonal antibody produced from rabbits immunized with synthetic peptide corresponding to amino-terminal residues of human -actin; final concentration 1/1