Pperk1 2

The following antibodies were used: pSmad2 (3108; Cell Signaling, Danvers, MA), Smad2/3 (SC-133098; Santa Cruz Biotechnology), ezrin (E8897; Sigma-Aldrich), ppERK1/2 (M9692; Sigma-Aldrich), ERK2 (SC-154; Santa Cruz Biotechnology), pMEK (9154; Cell Signaling), MEK (2352; Cell Signaling), and lamin A/C (SC-376248; Santa Cruz Biotechnology).

Cells grown in 96-well plates were fixed with –20°C 100% MeOH and incubated at 4°C overnight (endogenous) or 3.7% paraformaldehyde for 5 min (fluorescent protein–containing samples). Fixed cells were rehydrated in phosphate-buffered saline and blocked with 0.5% bovine serum albumin (BSA) in 50 mM Tris-HCl supplemented with 0.05% Tween-20 for 1 h. Primary antibody was substituted for BSA at a dilution of 1:200 for 3 h at room temperature. Secondary antibodies were used at a dilution of 1:200 for 1 h at room temperature. The antibodies used were ppERK1/2 (M7802; Sigma-Aldrich), E-cadherin (SC-21791; Santa Cruz Biotechnology, Dallas, TX), Alexa 555 goat anti-rabbit (Molecular Probes, Grand Island, NY), and Alexa 488 goat anti-mouse (Molecular Probes).