4.6. Western Blot Analysis

Cells (8 × 10⁵) were seeded into 60-mm dishes. For both pERK1/2 and pMLC (Phospho-Myosin Light Chain 2, Thr18/Ser19) immunoblots, cells were harvested 24 h after seeding by direct lysis in 2× Laemmli buffer mixed with equal volume of RIPA buffer (10 mM Tris-Cl pH 8.0, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl, protease/phosphatase inhibitor cocktail Thermo Fisher, Waltham, MA, USA, cat#88266). Samples were sonicated 2× for 5 s and heated for 5 min at 95 °C. Equal volumes of protein lysate (30 µL) for each cell line were resolved using 15% SDS-PAGE gels, transferred to PVDF membranes, and blocked in 5% BSA in Tween tris-buffered saline. Primary and secondary antibodies were diluted in 1% BSA and 0.01% Triton X-100 in PBS. Membranes were incubated in 1:1000 diluted primary antibodies pERK1/2 pT202/Y204 (Calbiochem, Burlington, MA, USA cat# KP26001), total ERK1/2 (Cell Signaling, Danvers, MA, USA, cat# 9102), pMLC (Cell Signaling, cat# 3674S), and GAPDH (Santa Cruz, cat#365062) overnight at 4 °C. HRP-conjugated anti-rabbit (Cell Signaling, cat# 7074P2), and HRP-conjugated anti-mouse (Cell Signaling, cat# 7076P2) antibodies were diluted 1:6000. Blots were developed using FemtoGlow™ Western chemiluminescent HRP substrate (Michigan Diagnostics, Royal Oak, MI, USA, cat# FWPS02). Bands were visualized and quantified using a Li-Cor Odyssey Fc scanner and quantified using Image Studio Lite Version 5.2. The full western blots were shown in Figures S4 and S5.