rabbit anti aha1  (StressMarq)

Journal: EMBO Reports

Article Title: SUMOylation of protein phosphatase 5 regulates phosphatase activity and substrate release

doi: 10.1038/s44319-024-00250-2

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Rabbit anti-Aha1 , StressMarq Biosciences , Cat# SPC-183; RRID:AB_1944037.

Techniques: Recombinant, Sequencing, Synthesized, Software, In Vitro

Journal: Communications Biology

Article Title: Benzbromarone interferes with the interaction between Hsp90 and Aha1 by interacting with both of them

doi: 10.1038/s42003-025-08189-3

Figure Lengend Snippet: A Chemical structure of Benzbromarone. B Ligand-observed CPMG spectra indicate that Benzbromarone could specifically bind to Hsp90-NTD and Aha1-CTD. The CPMG spectra are colored in red for Benzbromarone alone, and colored in cyan for Benzbromarone with the presence of either Hsp90-NTD or Aha1-CTD. C Isothermal titration calorimetry approach was applied to determine the thermodynamic parameters for the binding of Benzbromarone to Aha1-CTD. The K d value for Aha1-CTD:Benzbromarone system was calculated to be 0.84 ± 0.35 µM. D The dissociation constant for the binding of Benzbromarone to Hsp90-NTD was determined by the global fitting analysis of normalized resonance intensity changes (1-I/I 0 ) revealed by [ 1 H, 15 N] HSQC titration experiments. The K d value for Hsp90-NTD:Benzbromarone system was calculated to be 15.10 ± 3.86 µM.

Article Snippet: Anti-Aha1 (14725-1-AP) and Anti-α tubulin (66031-1-Ig) were purchased from Proteintech (Wuhan, China).

Techniques: Isothermal Titration Calorimetry, Binding Assay, Titration

Journal: Communications Biology

Article Title: Benzbromarone interferes with the interaction between Hsp90 and Aha1 by interacting with both of them

doi: 10.1038/s42003-025-08189-3

Figure Lengend Snippet: A The binding sites of Benzbromarone in Hsp90-NTD were characterized by carrying out [ 1 H, 15 N] HSQC NMR titration experiments. Superposition of [ 1 H, 15 N] HSQC spectra of Hsp90-NTD without (red) and with the presence of Benzbromarone (black, molar ratio of 1:1 for Hsp90-NTD to Benzbromarone) reveals spectral changes upon Benzbromarone’s binding. The residues in Hsp90-NTD that might be involved in the recognition of Benzbromarone were identified by performing chemical shift perturbation (CSP) analysis ( B ) and NMR signal intensity change analysis C . The mean and mean + S.D. CSP values for amino acid residues in Hsp90-NTD are represented by dashed line and solid line, respectively. The mean relative intensity change value for amino acid residues in Hsp90-NTD is represented by dashed line. Residues with chemical shift perturbations greater than mean + S.D. are labeled. Proline residues and those residues with fully attenuated resonances upon the addition of Benzbromarone are denoted by green dots and red dots, respectively. D Expanded view of the possible binding mode for Benzbromarone to Hsp90-NTD, which was revealed by molecular docking. Detailed interactions between Benzbromarone and its surrounding residues in Hsp90-NTD are shown, and intermolecular hydrogen bond details are highlighted. The PDB code for Hsp90-NTD:AMPPNP complex structure is 3T1K. E The binding sites of Benzbromarone in Aha1-CTD were characterized by [ 1 H, 15 N] HSQC NMR titration experiments. Superposition of [ 1 H, 15 N] HSQC spectra of Aha1-CTD without (red) and with the presence of Benzbromarone (black, molar ratio of 1:2 for Aha1-CTD to Benzbromarone) reveals spectral changes upon Benzbromarone’s binding. F The residues in Aha1-CTD that might be involved in the recognition of Benzbromarone were identified by performing chemical shift perturbation (CSP) analysis. The mean and mean + S.D. CSP values are represented by dashed line and solid line, respectively. Residues with chemical shift perturbations greater than mean + S.D. are labeled. Proline residues and those with significantly attenuated resonances upon Benzbromarone’s addition are denoted by green dots and red dots, respectively. G The crystal structure of Aha1-CTD complexed with Benzbromarone (PDB code: 8Z3J) reveals the binding mode of the compound to the protein. The expanded view of Benzbromarone’s binding pose to Aha1-CTD is depicted in the left panel, illustrating detailed interactions between Benzbromarone and its surrounding residues within Aha1-CTD, with intermolecular hydrogen bonds highlighted. For comparison, the right panel shows a superposition of the crystal structure of Aha1-CTD complexed with Benzbromarone (PDB code: 8Z3J, in gray) and the solution NMR structure of Aha1-CTD in its apo state (PDB code: 1X53, in light green). H The ribbon and the surface presentation for the crystal structure of Aha1-CTD in free state (PDB code: 8Z3H) are shown in the left and the right panel, respectively. I The ribbon and the surface presentation for the crystal structure of Aha1-CTD complexed with Benzbromarone (PDB code: 8Z3J) are shown in the left and the right panel, respectively.

Article Snippet: Anti-Aha1 (14725-1-AP) and Anti-α tubulin (66031-1-Ig) were purchased from Proteintech (Wuhan, China).

Techniques: Binding Assay, Titration, Labeling, Comparison

Journal: Communications Biology

Article Title: Benzbromarone interferes with the interaction between Hsp90 and Aha1 by interacting with both of them

doi: 10.1038/s42003-025-08189-3

Figure Lengend Snippet: A The results for [ 1 H, 15 N] HSQC-based competitive binding experiments are shown. Zoomed view of the superimposed [ 1 H, 15 N] HSQC spectra for 15 N-Aha1-CTD alone (red), 15 N-Aha1-CTD with the presence of Hsp90-NTD (with AMPPNP, blue, molar ratio of 1:5 for 15 N-Aha1-CTD to Hsp90-NTD), 15 N-Aha1-CTD with the presence of Hsp90-NTD and Benzbromarone (with AMPPNP, green, molar ratio of 1:5:8 for 15 N-Aha1-CTD to Hsp90-NTD to Benzbromarone), and 15 N-Aha1-CTD with the presence of Benzbromarone (purple, molar ratio of 1:8 for 15 N-Aha1-CTD to Benzbromarone). The directional changes in chemical shift perturbations induced by Hsp90-NTD or Benzbromarone binding are indicated by black arrow and red arrow, respectively. B The determined ATPase activity data of Hsp90β (solid circle) and Hsp90β:Aha1(solid triangle) upon the treatment of Benzbromarone are shown. The ATPase rates obtained from the NADH-coupled ATPase experiments were plotted, along with the mean ± SD derived from three times of independent repeats ( n = 3; each repeat contains one experimental data extracted from technical triplicates). Statistical significance was determined using unpaired t -test (*** p < 0.001, **** p < 0.0001). C The ATP hydrolysis reaction catalyzed by Hsp90β was monitored by collecting 1D 31 P NMR spectra. Overlay of 1D 31 P spectra of the Hsp90β:ATP (4 μM:1 mM) reaction system without (red) and with the addition of Benzbromarone (400 µM, green) acquired at the time points of 2 h and 4 h after the initiation of reaction, are shown. D The ATP hydrolysis reaction catalyzed by Hsp90β and Aha1 was monitored by collecting 1D 31 P spectra. Overlay of 1D 31 P spectra of the Hsp90β:Aha1:ATP (4 μM:20 μM:1 mM) reaction system without (red) and with the addition of Benzbromarone (400 µM, green) are presented. The spectra were acquired at the time points of 20 min and 1 h after the initiation of reaction.

Article Snippet: Anti-Aha1 (14725-1-AP) and Anti-α tubulin (66031-1-Ig) were purchased from Proteintech (Wuhan, China).

Techniques: Binding Assay, Activity Assay, Derivative Assay

Journal: Communications Biology

Article Title: Benzbromarone interferes with the interaction between Hsp90 and Aha1 by interacting with both of them

doi: 10.1038/s42003-025-08189-3

Figure Lengend Snippet: A Cellular thermal shift assay was applied to detect the binding of Benzbromarone to Hsp90 and Aha1 in cellular context. Upon the treatment of Benzbromarone, the cellular thermal stabilities of Hsp90 and Aha1 were up-regulated, and the positive shifting for the temperature-dependent melting of both Hsp90 and Aha1 was observed. The cellular thermal shift experiments were all repeated for three times. Raw blot data from multiple runs ( n = 3) were processed by ImageJ and graphs were plotted with GraphPad Prism 8. The quantitative analysis plots for Hsp90 and Aha1 are shown in the middle panel and the right panel, respectively. B Cell viability of LNCaP, MNNG/HOS Cl #5, MG-63, LS174T, HCT116 and HT-29 cells were assessed after their exposure to vehicle and different concentrations of Benzbromarone for 72 h. Data were analyzed by GraphPad Prism 8 and is presented as means ± standard deviation ( n = 3). The IC 50 values of Benzbromarone against LNCaP, MNNG/HOS Cl #5, MG-63, LS174T, HCT116 and HT-29 cells were determined to be 44.05 ± 2.93 µM, 45.54 ± 3.47 µM, 69.15 ± 3.59 µM, 76.28 ± 6.79 µM, 55.93 ± 1.82 µM and 59.62 ± 3.73 µM, respectively. (C) Colony formation experiments were performed by using LNCaP, MNNG/HOS Cl #5, MG-63, LS174T, HCT116 and HT-29 cells treated with different concentrations of Benzbromarone (30 µM for LNCaP, LS174T, HCT116 and HT-29 cells, 20 µM for MNNG/HOS Cl #5 cells, 25 µM for MG-63 cells), and the quantitative results are presented in lower panel ( n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, t -test).

Article Snippet: Anti-Aha1 (14725-1-AP) and Anti-α tubulin (66031-1-Ig) were purchased from Proteintech (Wuhan, China).

Techniques: Thermal Shift Assay, Binding Assay, Standard Deviation

Journal: Communications Biology

Article Title: Benzbromarone interferes with the interaction between Hsp90 and Aha1 by interacting with both of them

doi: 10.1038/s42003-025-08189-3

Figure Lengend Snippet: Benzbromarone suppresses tumor cell growth and metastasis through dual mechanisms: competitively inhibiting Hsp90 ATPase activity and interfering with Hsp90-Aha1 interactions.

Article Snippet: Anti-Aha1 (14725-1-AP) and Anti-α tubulin (66031-1-Ig) were purchased from Proteintech (Wuhan, China).

Techniques: Activity Assay