Eea1 antibody

For dextran uptake, cells with or without treatment were incubated with 100 μg/ml Dextran-568, or Dextran-488 (10,000 MW, Anionic, Fixable, ThermoFisher Scientific) for 30 min at 37 °C. After washing with PBS, cells were fixed with 4% paraformaldehyde (PFA), washed with PBS, and stained for actin with Phalloidin-488 (Thermo Fisher Scientific, A12379), for 20 min at room temperature. After washing with PBS for three times, cells were dried in dark, and mounted with ProLong® Gold Antifade Mountant (Thermo Fisher Scientific, P36930) before imaging with Zeiss LSM confocal microscope.
Immunofluorescent analysis has been previously described by us (Das et al., 2015 (link); Yu, Nie, et al., 2014; (link)
Yu, Yehia, et al., 2014 (link)). Briefly, cells with or without treatment were washed with PBS twice and fixed with 4% PFA at room temperature for 15 min. Cells were then washed with PBS and blocked with 10% donkey serum in PBS with 0.1% Triton X-100 for 1 hr at room temperature. Cells were incubated with EEA1 antibody (Cell Signaling Technology, Beverly, MA, #3288), at 1:200 dilution in blocking buffer over-night at 4 °C. Cells were washed with PBS and incubated with Alexa-555 conjugated anti-rabbit secondary antibody (1:500) and Phalloidin-Alexa-488 (1:3000) at room temperature for 1 hr before washing and mounting.

Immunocytochemistry was performed using antibodies to EMT markers that included E-cadherin and vimentin (Millipore, Burlington, MA, USA), N-cadherin (R&D Systems, Minneapolis, MN, USA), metalloproteinase 9 (MMP9) and P120 (Abcam, Cambridge, MA, USA), and active (non-phosphorylated) β-catenin (Cell Signaling, Danvers, MA, USA). Also, an early endosome antigen 1 (EEA1) antibody (Cell Signaling, Danvers, MA, USA) was used for the E-cadherin colocalization study. Briefly, cells were grown in Ibidi chamber slides (Ibidi, Munich, Germany) and fixed using 4% paraformaldehyde. Cells were blocked in 10% donkey serum (Sigma-Aldrich, St. Louis, MO, USA) in 0.1% Triton-X (Bio-Rad, Hercules, CA, USA) in PBS, and primary antibodies were diluted in 0.2% Tween (Sigma-Aldrich, St. Louis, MO, USA) in PBS and incubated at 4°C overnight. Cells were next washed in PBS then incubated in Alexa Fluor 594 and 488 secondary antibodies (Life Technologies, Eugene OR USA) at room temperature for 1 hour, then mounted in Vectashield (Vector Laboratories, Burlingame, CA USA).