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. 2017 May 24;8:15328. doi: 10.1038/ncomms15328

Figure 7. A107N restores function and conformation of Hsp82-Y606E.

Figure 7

See also Supplementary Fig. 6. (a) hsc82hsp82 cells expressing Hsp82 WT (MR923), Y606E (CLY24), A107N (AF3) and A107N-Y606E (MR1016) were transformed pG/N795-GR and pUCΔSS-26X. Resultant colonies were grown to mid-log phase followed by the addition of synthetic hormone. Following a 5-h incubation, β-galactosidase activity was quantified (each sample was run in triplicate and error bars represent s.d. from the mean). These same strains were lysed and analysed using SDS–PAGE and the indicated antibodies. (b,c) hsc82hsp82 cells were transformed with the indicated His-tagged Hsp82 constructs. Resultant colonies were grown overnight in rich media and cell pellets were collected. Collected cell pellets were lysed and His-complexes were isolated using nickel resin in the presence (+) or absence (−) of AMP-PNP. Following SDS–PAGE, His-complexes were analysed by Western blot as in Fig. 6.