K lisa mtor activity kit

The activity of mTOR in both cell-free and cell-based systems was measured with colorimetric K-LISA mTOR activity kit (EMD Millipore, USA). In the cell-based assay, whole cell lysates were prepared from RPMI-8226 cells followed by co-immunoprecipitation with an anti-mTOR antibody. The elutes were then subject to clioquinol or Rapamycin treatment for 20 min, followed by incubation with adenosine triphosphate. In the cell-free system, recombinant mTOR kinase was first treated with clioquinol or wortmannin for 20 min, followed by incubation with adenosine triphosphate in the glutathione-coated 96-well plate where mTOR phosphorylates P70S6K at Thr389. The phosphorylated substrate was detected with anti-P70S6K (Thr389) antibody, followed by detection with HRP-antibody conjugate and TMB substrate (3,3′,5,5′,-tetramethylbenzidina). Relative activity of mTOR kinase was determined by reading the absorbance at the wavelength of 450 nm.

For measurement of the influence of macrolides on mTOR activity, the K-LISA mTOR Activity Kit (Merck Millipore, Darmstadt, Germany) was used according to the manufacturers' instructions. In short, the indicated concentrations of AZM or 500 nM RAPA were pre-incubated on ice in the presence or absence of recombinant FKBP12 (3 μg per sample) for 20 minutes. Plain buffer and FKBP12 without antibiotics served as negative and positive control respectively. A recombinant mTOR fragment (amino acids 1360–2549, 50 ng per sample) containing the kinase domain was added for another 20 minutes. Afterwards, samples were transferred to microtiter plates pre-coated with a p70S6K-GST fusion protein (500 ng per well) and incubated at 30°C for 30 minutes. Subsequently, mTOR-mediated phosphorylation of p70S6K was determined in an ELISA using a monoclonal antibody specific for p70S6K phosphorylated at Thr389.
Effects of AZM on the PI3-K were examined using the PI3-kinase activity ELISA (Echelon, Salt Lake City, UT). In short, the indicated concentrations of AZM were added to 50 ng recombinant PI3-K (p110α/p85α; Echelon) and conversion of PIP₂ substrate to PIP₃ was performed for 2 hours at 37°C. Afterwards, concentrations of PIP₃ were determined using a competitive ELISA system.