Soybean trypsin inhibitor

Control, Atg5^−/−, and Atg7^−/− MEFs purchased from Riken Cell Bank (Tsukuba, Japan) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS, Sigma-Aldrich) and 1% penicillin and streptomycin (GIBCO) at 37°C with 5% CO₂ (Kuma et al., 2004 (link)). For the adhesion assay (Hu et al., 2008 (link)), the Rho activation assay (Ren et al., 2000 (link)) and western blotting (Cheng et al., 2014 (link)), cells were grown to 60% confluence and trypsinized with 0.05% tripsin-EDTA (Gibco, Thermo Fisher Scientific, MA, USA). The trypsinization was stopped by addition of 0.5 mg/ml soybean trypsin inhibitor (Wako, Osaka, Japan) in DMEM. The cells were pelleted and washed once more with 0.5 mg/ml soybean trypsin inhibitor, followed by another wash with serum-free medium, then were suspended in DMEM containing 0.1% bovine serum albumin (BSA, Sigma) and maintained in suspension for 1 h at 37°C (Cheng et al., 2014 (link)). The suspended cells (2×10⁵ cells/ml) were plated on collagen I–coated dishes (Corning, NY, USA) and incubated at 37°C. EGFP-m-Paxillin (Plasmid #80023) was obtained from Addgene. We transfected 1 µg plasmid into 2×10⁴ MEFs using Lipofectamine® 2000 reagent (Thermo Fisher Scientific) according to the manufacturer's protocol.