The Leica TCS SP8 confocal microscope was equipped with an environment-controlled chamber (Life Imaging Services) maintained at 37°C and an HC PL APO CS2 1.4 NA oil objective (Leica Microsystems). Alexa Fluor 488–conjugated secondary antibodies, fluorescent fusion proteins containing mCitrine and mCherry, and EGF–Alexa Fluor 647 were excited using a 470- to 670-nm white light laser (Kit WLL2, NKT Photonics) at 488, 514, 561, and 647 nm, respectively. PH-Akt-Cerulean was excited using an argon laser at 458 nm. Detection of fluorescence emission was restricted with an AOBS as follows: Cerulean at 468 to 505 nm, Alexa Fluor 488 at 498 to 551 nm, mCitrine at 525 to 570 nm, mCherry at 570 to 650 nm, and Alexa Fluor 647 at 654 to 754 nm. The pinhole was set to 250 μm, and 12-bit images of 512 × 512 pixels were acquired in a frame-by-frame sequential mode.

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