Normal mice igg

EGF was purchased from American R&D Company (Minneapolis, MN, United States). Cycloheximide (CHX) was purchased from Sigma-Aldrich Corporation (United States). Anti-human DENND1A monoclonal antibody, anti-human Grb2 monoclonal antibody, anti-human HA monoclonal antibody and anti-EGFR monoclonal antibody were purchased from Cell Signaling Technology (Danvers, MA, United States). Normal mice IgG, normal rabbit IgG and anti-GAPDH polyclonal antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States). Anti-GST antibody was purchased from cwbiotech (Beijing, China) and anti-Rab35 polyclonal antibody was purchased from Biogot Technology (Nanjing, Jiangsu, China). Anti-Flag monoclonal antibody and FITC-labeled secondary antibody were purchased from Sigma-Aldrich Corporation. TRITC-labeled and horseradish peroxidase (HRP)-labeled secondary antibodies were purchased from Jackson ImmunoResearch (United States).

The A549i cells were inoculated into eight 150-mm plates, and Dox was added to the final concentration of 1 μg/mL when the cell concentration reached 70–80%. After 2 days of culture, the cells were cross-linked with 1% formaldehyde (final concentration) and sonicated with the following parameters: 30 s on, 30 s off, 25% set power for 15 cycles. The total Lysis was divided into two bisected samples, one mixed with 1 mg/mL of Flag antibody and 40 μL of protein agarose bead. The other was mixed with 1 mg/mL of normal mice IgG (#F2416, Santa Cruz Biotechnology) and 40 μL of agarose beads. After overnight incubation, the beads were washed with a ChIP cleaning buffer. Chromatin was eluted, cross-linked DNA was reversed and DNA was extracted with phenol/chloroform. Finally, the eluted DNA was resolved in ddH2O and then sent to BGI (https://www.bgi.com/bgi-online) for sequencing.