Analysis of RNP formation at different temperatures. Infected 293T cells were incubated at 37°C or 41°C for 5 h and lysed as described in Methods. The extracts were layered onto 5–20% sucrose gradients and separated by centrifugation. The resulting fractions were analysed by Western blotting to detect (a, b) NP and (c, d) PB1. (e-h) RNA was also extracted from each fraction and subjected to primer extension analysis to detect segment 7-specific RNA.