Homogeneous time resolved fluorescence (HTRF) assay.
The HTRF assays were performed similarly to those previously described
(43 ). The antibody pairs concentrations is 0.023/1.4 µg/mL (in
HTRF buffer with 1% Triton X-100) were optimized for different antibody
pairs and cell lines in different experiments. For human fibroblasts
cells and human iPS-derived neurons, the antibody pair is 2B7/MW1, For
mouse STHdh cells and mouse brain tissue, the antibody is 2B7/2166. For
all the experiments, the protein concentration (by BCA, Pierce, cat. no.
23225) was measured to ensure equal loading or cell viability. Different
protein concentrations or cell numbers per well were tested to ensure
that the signals were in the linear range. Background corrections were
performed by subtracting the background signals from blank samples.