Odyssey clx imager system

HEK-293 cells were infected with rChAd63-mDS-Cav1. At 48 h after infection, the cells were collected, washed twice with cold PBS, and blotted onto nitrocellulose membranes. The antibodies of palivizumab, 131-2A and D25 specific for epitopes of site II, site I and site Ø in F, respectively, were constructed and prepared by cloning the genes encoding heavy (VH) and light (VL) chain, respectively, into expression vectors, and were employed to incubation with the above samples. After further incubated with IRDye 800CW goat anti-human IgG or anti-mouse IgG (Li-Cor Biosciences, Lincoln, NE, USA), these samples were finally visualized by the LI-COR Odyssey Clx Imager system (Li-Cor Biosciences).

The thioesterase reaction conditions were chosen based on previously published methodologies [15 (link),16 (link)]. RpfF thioesterase activity was measured using a 10-μL reaction consisting of 78 μM C12:0–ACP_Ec substrate, 0.64 μM RpfF_Bc (or control buffer N), and either 6.4 μM RpfR_Bc(FI), 1.28 μM RpfR_Bc(FI), or control buffer D in the thioesterase assay buffer (100 mM Tris [pH 7.5]). This reaction was incubated at 37 °C for 30 min and then heat inactivated at 95 °C for 2 min. Reactions were analyzed with a conformation-sensitive nondenaturing gel containing 20% polyacrylamide, 375 mM Tris (pH 8.8), and 2.5 M urea. Gels were stained with Coomassie Brilliant Blue, and band intensities were measured using a LI-COR Odyssey CLx Imager System (LI-COR Biosciences) and quantified using Image Studio version 3.1 (LI-COR Biosciences).

Tissue collection and western blot were performed as described previously [17,44,45]. Briefly, 40 μg of 10% nasal turbinate homogenates was used for the analysis of PrP^C (D18 at 1:5000, produced in house), GAP43 (rabbit-anti-GAP43 antibody at 0.4 μg/μl) and OMP (goat-anti-OMP polyclonal antibody, Wako Chemicals, Richmond, VA at a 1:5000 dilution). For detection of GAPDH loading control, chicken-anti-GAPDH antibody (EMD Millipore) was used at 0.33 μg/μl. The secondary antibodies were IRDye conjugates available from LI-COR (Lincoln, NE): 800CW Gt Anti-Human IgG for detection of D18, 800CW Dky Anti-Goat IgG, 680RD Dky Anti-Rabbit IgG, and 680RD Dky Anti-Chicken IgG, all at concentrations of 0.02 μg/ml. Blots were visualized on the Odyssey CLx Imager system (LI-COR) and associated Image Studio 4.0 software was used to measure the total signals of each protein. For PrP^C analysis, signal levels were log-transformed for comparison purposes and normalized to the GAPDH signal within each lane. Quantification of the relative differentiation status was performed by comparing the GAP43:OMP signal ratio of methimazole treated samples to the vehicle for each time point. Statistical analysis was performed using Student’s t-test (vehicle:MTZ), with P values of <0.05 considered significant.