Texas red conjugated egf

To monitor EGFR traffic, HeLa cells stably expressing EGFR-EGFP-3×FLAG were serum-starved in DMEM supplemented with 0.5% FBS for 24 h, followed by 2 h in 0% FBS/DMEM. Subsequently, cells were stimulated with 100 ng ml⁻¹ of Texas Red-conjugated EGF (Thermo Scientific) at 37 °C for the indicated times. Cells were fixed in 3% formaldehyde/PBS for 15 min on ice, permeabilized with 0.5% Triton X-100/PBS for 10 min, and blocked with 10% FBS/PBS. Cells were subsequently incubated with anti-EEA1 antibody (1 μg ml⁻¹; #E41120, BD Transduction Laboratories) in blocking buffer for 1 h at room temperature, washed three times with PBS, and incubated with Alexa Fluor 647-conjugated anti-mouse IgG antibody (1:1000; Invitrogen) in blocking buffer for 1 h at room temperature. Nuclei were stained with 4′,6-diamidino-2-phenylindole. Fluorescence images were captured on a LSM710 laser-scanning confocal microscope (Carl Zeiss) equipped with a PLAN/APO ×63 oil objective and the ZEISS ZEN 2011 software.

HeLa cells (laboratory stock of M. Komada)^{46 (link)} were grown in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin in a humidified 37 °C incubator with 5% CO₂. To introduce EGFR-EGFP-3×FLAG to the AAVS1 locus in HeLa cells, pTALEN-AAVS1 (left), pTALEN-AAVS1 (right), and pZDoner-EGFR-EGFP-3×FLAG expression plasmids were co-transfected into HeLa cells using a NEPA21 Super Electroporator (NEPAGENE). Cells were maintained in DMEM supplemented with 10% FBS and penicillin/streptomycin. After 2 days, culture medium was replaced with DMEM supplemented with 10% FBS, 0.5 mg ml⁻¹ G418, and penicillin/streptomycin. Cells were maintained for 3–5 days, and clones that expressed the protein were selected under a confocal microscope and maintained for another 1–2 weeks. Media containing G418 were replaced every 2 days. For stimulation with EGF, cells were serum-starved in DMEM supplemented with 0.5% FBS for 24 h, followed by 2 h in 0% FBS/DMEM. Cells were subsequently incubated with human EGF (100 ng ml⁻¹; PeproTech, Rocky Hill, NJ) or Texas Red-conjugated EGF (100 ng ml⁻¹; Thermo Scientific) at 37 °C.