Irdye conjugates

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.

30 embryos injected with RNA were homogenized in 300 µl lysis buffer (20 mM Tris/HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mM PMSF) and incubated on ice for 5 min. Protein samples were cleared at 13,000 rpm at 4°C for 5 min. For removal of lipids, the supernatant was mixed with 50 µl Freon followed by centrifugation at 13,000 rpm at 4°C for 5 min. The upper protein containing phase was collected. Concentration of protein samples was determined by Bradford assay with BSA as standard. Western blotting was performed according to standard procedures and proteins were visualized using a Li-COR ODYSSEY Imager. Primary antibodies were purchased from Abcam (rabbit polyclonal Mef2C, directed against residues 450 to the C-terminus of human MEF2C), Santa Cruz (mouse monoclonal anti Mef2D, H11, epitope mapping within an internal region of MEF-2D of human origin) and Serotec (γ-Tubulin, clone YL1/2). Secondary antibodies used were IRDye conjugates from Li-COR.