Journal: Science Advances

Article Title: The immune-evasive proline-283 substitution in influenza nucleoprotein increases aggregation propensity without altering the native structure

doi: 10.1126/sciadv.adl6144

Figure Lengend Snippet: ( A ) Representative SYPRO Orange thermal unfolding curve of Pro 283 NP, Ser 283 NP, and SYPRO Orange dye with buffer only. Means of four technical replicates are shown with the shaded area representing the SD. ( B ) Circular dichroism spectra of Pro 283 and Ser 283 NP. ( C ) Irreversible T agg was calculated by fitting the plots from (A) to a Boltzmann sigmoidal curve. The experiment was performed at three different NP concentrations. Individual measurements for five technical replicates are shown (**** P < 0.0001). ( D ) Schematic for a cellular thermal shift assay experiment. Intact cells ectopically expressing NP are harvested, split into aliquots, and heated rapidly to the indicated temperatures in a thermocycler. After centrifugation to pellet protein aggregates, soluble NP remaining in the supernatant is quantified via immunoblot. ( E ) Quantification of cellular thermal shift assay immunoblots. The mean of biological triplicates is shown with the error bars representing the SD (* P < 0.05). All band intensities were normalized to 37°C. Original blots are shown in fig. S2.

Article Snippet: For differential scanning fluorimetry, T agg values were obtained by performing a nonlinear regression to a Boltzmann sigmoid function using GraphPad Prism.

Techniques: Circular Dichroism, Thermal Shift Assay, Expressing, Centrifugation, Western Blot