FIG. 1.

Stable cell lines with tetracycline-regulated expression of hsc70, hsp70, or hsp70 domain mutant proteins. (A) Schematic representation of hsp70 functional domains. hsp70 and hsc70 have an amino-terminal ATPase domain followed by a peptide binding domain and the C-terminal sequence EEVD. (B) Flow-cytometric profiles of GFP expression in each of the clones grown in the presence (shaded profile) and absence (open profile) of doxycycline. The expression cassette is dicistronic and carries both the gene of interest and the GFP gene. In the BFP-expressing cell line the dicistronic transcript carries both the BFP and GFP genes. All of the cell lines were induced for 24 h except for the hsc70 and BFP-expressing cell lines, which were induced for 48 h. (C) Western blot analysis showing the levels of expression of the proteins encoded by the first cistron. Cells were grown with or without doxycycline as described for panel B. The upper portion shows the results with antibody N27, which recognizes both hsc70 and hsp70. The bottom portion shows the results for hsp70-specific antibody C92. Purified hsp70 (25 to 400 ng) was included to insure linearity of the signals.