Figure 3.

Heat shock induces insolubilization of eIF4G. 293T cells were maintained at 37°C or heat shocked at 44°C for the times shown. (A) Cell extracts were prepared using nonionic detergent (Triton X-100) or ionic detergent (SDS at 100°C) to solubilize proteins, equal amounts were resolved by SDS-PAGE and immunoblotted for eIF4G, eIF4E, eIF4A, eIF4B, eIF3, or PABP. Four subunits of eIF3 are shown. (B) Cells were labeled in vivo with ³²PO₄ during the time course of heat shock, eIF4E was recovered by immunoprecipitation and analyzed as described in the legend to Figure 2. (C) Cells were labeled in vivo with 50 μCi/ml of [³⁵S]methionine for 30 min during heat shock as shown, equal amounts of protein extracts were resolved by SDS-PAGE and fluorographed. Results are typical of at least three independent experiments and were quantitated by digital densitometry.