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. Author manuscript; available in PMC: 2016 May 31.
Published in final edited form as: Cell Rep. 2016 Jan 21;14(4):872–884. doi: 10.1016/j.celrep.2015.12.084

Figure 2. Impact of T101-yHsp90 and T115-hHsp90α Phosphorylation on ATPase Activity and Co-chaperones Binding.

Figure 2

(A) Relative ATPase activity of WT yHsp90-His6 and yMps1 mediated phospho-T101-yHsp90-His6. ATPase activity was inhibited by 10 μM GB. Error bars represent SD of three independent experiments. ****p < 0.0001.

(B) Relative ATPase activity of WT hHsp90α-His6 and hMps1 mediated phospho-T115-hHsp90α-His6. ATPase activity was inhibited by 10 μM GB. Error bars represent SD of three independent experiments. ****p < 0.0001.

(C) yHsp90 was isolated from yeast with empty plasmid (C), WT yHsp90-His6 (WT), T101A-yHsp90-His6 (T101A), and T101E-yHsp90-His6 (T101E). Interaction of the co-chaperones yAha1, Cdc37p50, Sba1p23, and Sti1Hop were detected by immunoblotting. Equivalent precipitation of yHsp90 was confirmed by blotting for hexahistidine.

(D) HEK293 cells were transfected with empty plasmid (C), hHsp90α-FLAG (WT), T115A-hHsp90α-FLAG (T115A), or T115E-hHsp90α-FLAG (T115E). Hsp90-FLAG was immunoprecipitated (IP) with anti-FLAG M2 affinity gel and interaction with hAha1 or p50cdc37, p23Sba1, or p60Hop was examined by immunoblotting.