Glass bottom dishes

Cells expressing either fluorescent protein-tagged or epitopetagged proteins were plated on 35-mm-diameter glass-bottom dishes (AGC Techno Glass, Shizuoka, Japan) coated with poly-Llysine-(for hematopoietic cells) or collagen-coated (for nonhema-topoietic cells). During the observation, cells were maintained in phenol red-free RPMI 1640 (for hematopoietic cells, Thermo Fisher Scientific) or phenol red-free DMEM/F12 (Thermo Fisher Scientific).
For imaging of FRET, cells were subjected to time-lapse, dualemission microscopy with an interval of every 30 sec. Beginning at 10 min, cells were treated with 2 μM imatinib. To represent FRET efficacy, the FRET/CFP emission ratio was calculated as the quotient of background-subtracted FRET and CFP images and normalized to that at time 0. In Fig. 3, FRET/CFP ratio images are shown in intensity-modulated display mode, in which eight colors from red to blue represent the normalized emission ratios, with the intensity of each color indicating the mean intensity of FRET.

The expression vectors in mammalian cells, pAcGFP1-Actin, pAcGFP1-Tubulin, and pDsRed-Monomer-C1, were obtained from Clontech. Oligonucleotides used as PCR primers in the construction of expression plasmids for fusion proteins were custom-synthesized by Gene Design (Osaka, Japan). The WST-1 cell proliferation assay system was purchased from Takara Bio (Kyoto, Japan). Lipofectamine 2000 was obtained from Invitrogen (Carlsbad, CA). Glass-bottom dishes used for cell culture and imaging experiments were purchased from AGC Techno Glass (Shizuoka, Japan).