An expression vector for green fluorescent protein-tagged epidermal growth factor receptor (EGFR-GFP), pEGFR-GFP, was constructed as described previously49 (link). In this vector, a monomeric mutation was introduced into the GFP-coding region (A206K) to prevent self-association50 (link). pEGFR-GFP was transiently transfected into MEFs grown on glass coverslips using Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. After incubation overnight, the MEFs were treated with MMC or fixed with 2.5, 5, 10, or 14% FA as described above. Immediately before experimental observations, the solution was replaced with minimum essential medium (Nissui, Tokyo, Japan) containing 1% bovine serum albumin and 5 mM Pipes (pH 7.2).
Single molecules of EGFR-GFP were observed by TIRF microscopy using a Nikon TE2000 inverted fluorescence microscope equipped with a 60 × NA 1.49 objective lens (Plan Apo, Nikon, Tokyo, Japan). The specimens were exposed to a 488-nm wavelength laser, and fluorescence images were acquired using an EM-CCD camera (Image EM; Hamamatsu Photonics, Hamamatsu, Japan) at a temporal resolution of 30.5 ms. All the single-molecule experiments were performed at 25 °C.
Single molecules of EGFR-GFP were observed by TIRF microscopy using a Nikon TE2000 inverted fluorescence microscope equipped with a 60 × NA 1.49 objective lens (Plan Apo, Nikon, Tokyo, Japan). The specimens were exposed to a 488-nm wavelength laser, and fluorescence images were acquired using an EM-CCD camera (Image EM; Hamamatsu Photonics, Hamamatsu, Japan) at a temporal resolution of 30.5 ms. All the single-molecule experiments were performed at 25 °C.