Anti her2 647

Cells were collected via EDTA-PBS (0.4% EDTA) treatment on ice. After washing with PBS, cells were blocked with PBS containing 2% bovine serum albumin, 5% normal goat serum, and 5 mM EDTA on ice for 30 min. Cells were incubated with the primary antibody: anti-HER2-647 (1:100; Biolegend, San Diego, CA), anti-pHER2 (1:200; Cell Signaling Technology, Beverly, MA) on ice for 30 min, washed with PBS, and then treated with the Alexa-555 secondary antibody to pHER2 (1:400, I Invitrogen, Carlsbad, CA) on ice for 15 min. After two PBS washes, the level of pHER2 and HER2 were measured with a FACSCalibur (Becton-Dickinson, San Jose, CA).

For MEMA, cells on the slides were fixed in methanol/acetone (1:1) at −20 °C for 20 min, whereas for coverslip-based experiments, cells were fixed in paraformaldehyde (4%) at room temperature for 10 min. Cells were then blocked with PBS, 5 % normal goat serum, and 0.1% Triton X-100 at room temperature for 30 min, and then incubated with primary antibodies: anti-HER2-647 (1:100; Biolegend), anti-pHER2 (1:100; Cell Signaling Technology) overnight at 4 °C. pHER2 primary antibodies was visualized with fluorescent Alexa-568 secondary antibodies raised in goats (1:500; Invitrogen) together with Hoechst 33342 (1:2000; Invitrogen), incubated at room temperature for 2 hr. Images were acquired with an epifluorescence microscope. Image analyses were conducted with CellProfiler. For quantification of YAP/TAZ localization, the ratios of median fluorescence intensity in the cytoplasmic (C) and nuclear (N) compartments of segmented cells were used. The cutoffs ratios were used to establish three classes: C > N (X < 1), N = C (1 < X < 1.2) and N > C (X > 1.2)