FIG. 5.

Absence of effect of PIMT overexpression on synthesis and levels of DnaK. A) Steady-state DnaK levels. The amount of DnaK in crude extracts from PIMT-overproducing and control strains was measured after electrophoresis on 11% polyacrylamide gels and immunoblotting with anti-DnaK antibodies. Lanes 1 and 2 were loaded with 2 μg of crude extracts from control and PIMT-overproducing cells grown at 37°C; lanes 3 and 4 were loaded with 2 and 4 μg, respectively, of crude extracts from control cells grown at 37°C and transferred to 43°C for 2 h; lanes 5 and 6 were loaded with 2 and 4 μg, respectively, of crude extracts from PIMT-overproducing cells grown at 37°C and transferred to 43°C for 2 h. B) DnaK synthesis. Cells (PIMT overproducing [squares] or control [circles]) growing exponentially in glycerol medium at 30°C and then transferred to 43°C were pulse-labeled for 2 min with [³⁵S]methionine at the indicated times, chased with nonradioactive methionine for 1 min, and kept on ice before lysis and immunoprecipitation of the crude extract with anti-DnaK antibodies. Proteins were resolved by electrophoresis, detected, and quantified by using a Molecular Dynamics PhosphorImager.