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. 2019 Jun 12;10:2574. doi: 10.1038/s41467-019-10463-y

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© This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2019

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Fig. 1 — Y313 phosphorylation-promoted Hsp90–Aha1 interaction is mediated by Aha1-C. a Hsp90-associated Aha1 is phosphorylated on Y313 to a higher level compared to the general Hsp90 pool. Hsp90 was immunoprecipitated from transfected 293A cells with specific antibodies (H90-10 or AC88) or co-precipitated with FLAG-tagged Aha1. Hsp90 protein levels were examined by western blot, and Y313 phosphorylation was detected with an antibody specific for the phosphorylation of this site. b Y313 phosphomimetic substitution increases Hsp90 interaction with Aha1 but decreases the interaction with p60^Hop. FLAG-tagged Hsp90 was immunoprecipitated from transfected cells. Co-precipitated endogenous Aha1, p23, and p60^Hop were detected by western blot. Precipitated Hsp90 proteins were stained with Coomassie Blue (CBB). c Y313E substitution decreases Hsp90 association with p60^Hop. FLAG-p60^Hop was co-expressed with HA-Hsp90 and was immunoprecipitated from cells. Hsp90 and p60^Hop were detected by western blot with indicated antibodies. d Y313E phosphomimetic substitution in Hsp90 increases association of wild-type Aha1 and Aha1-C but not Aha1-N. FLAG-tagged Aha1 (wild-type or individual domains) were co-expressed with HA-tagged Hsp90, and complexes were immunoprecipitated with anti-FLAG resin. Exogenous Hsp90 was detected by western blot with anti-HA antibody. E = glutamic acid, F = phenylalanine. Bottom: schematic illustration of the Aha1 protein. e Point mutations in Aha1-C substantially decrease association with Hsp90-Y313E. Experiments were performed as in d. Endogenous co-precipitated Hsp90 (wild-type) was discerned with the antibody SPS-771, which significantly favors recognition of untagged Hsp90. Bottom: Schematic illustration of Aha1 with point-mutations indicated. f K273A mutation in Aha1-C abolishes the stimulatory effect of Y313 phosphomimetic substitution on Aha1 association. Experiments were performed as in d. Please see Supplementary Fig. 1 for more information. Source data for this figure are provided as a Source Data File