Htrf transcreener adp kit

The synthetic routes of target compounds and their identification results are shown in the supporting information files. The compounds used in the biological assay were in DMSO with a stock concentration of 10 mM, and the maximum concentration of DMSO used was no more than 0.1%. All of the chemical reagents and biological reagents, including DMSO, were purchased from Sigma-Aldrich (St Louis, MO, USA). All cell lines used in study were purchased from Typical Culture Preservation Commission Cell Bank, Chinese Academy of Sciences (NCB). The antibodies used from Cell Signaling Technology (Beverly, MA, USA) were the PARP antibody (9542L), cleaved-PARP antibody ⁵⁶²⁵, anti-Caspase 3 antibody ⁹⁶⁶⁴, cleaved caspase-3 antibody ⁹⁶⁶⁴, Bcl-2 antibody ³⁴⁹⁸, Bax antibody ²⁷⁷², c-RAF antibody ⁹⁴²², Akt antibody (9272S), Hsp70 antibody ⁴⁸⁷⁶, Erk1/2 antibody ⁴³⁷⁰, and HER-2/ErbB2 antibody ²²⁴². The HIF-1a antibody (ab51608) was purchased from Abcam (Cambridge, MA, USA). The β-actin antibody (60008-1-Ig) was from the Proteintech Group (Wuhan Sanying Biotechnology, Hubei, China). Cisbio Bioassays in France supplied the HTRF transcreener ADP kit. Adriamycin (S1208) was purchased from Selleck (Texas, USA).

The HTRF transcreener ADP kit was purchased from Cisbio Bioassays. For the inhibition experiment, Hsp90, ATP and compounds were prepared in the enzymatic buffer provided with the kit and supplemented with 10 mM MgCl₂. Hsp90 protein at 200 nM was first pre-incubated at 37 °C for 1.5 h in the presence of various concentrations of compound. Then, ATP (100 μM) and HTRF detection reagents were added, and the plate was incubated at room temperature for 30 min. The fluorescence was then measured in an Analyst plate reader (Molecular Devices SpectraMax Paradigm; Ex 620 nM, Em 665 nM). The IC₅₀ was calculated by a four-parameter curve using GraphPad Prism.

CPUY201112 (prepared in our laboratory) and 17-DMAG (Selleck, TX, USA) were dissolved in DMSO (Sigma, St. Louis, MO, USA) and stored in aliquots at −20 °C for no more than one month before use. The vehicle (DMSO) was used as a control in experiments at a maximum concentration of 0.1%. The system for the detection of immunoblotted proteins was from LI-COR (Odyssey, NE, USA). All other chemicals and biochemistry reagents were obtained from Sigma-Aldrich (St Louis, MO, USA). The following antibodies were used at appropriate concentrations as recommended by the manufacturer: anti-poly (ADP-ribose) polymerase (PARP) (# 9542), anti-AKT (# 9272s), anti-c-RAF (# 9422), anti-phospho AKT (# 9275s), anti-Hsp90 (# 4874), anti-Hsp70 (# 4876), anti-Erk (# 4370), anti-p21 (# 2947), and anti-HER-2/ErbB2 (# 2242) were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-p53 (OP43) and anti-MDM2 (07–575) were purchased from Merck Millipore (Billerica, MA, USA). Anti-MDMX (ab16058) was from Abcam (Cambridge, MA, USA). β-actin antibodies was purchased from LSBio (Seattle, WA, USA). The HTRF transcreener ADP kit was from Cisbio Bioassays (BP, Codolet, France).