Protocols for immunoblotting were as described previously (Inubushi et al., 2018 (link)). Briefly, cells were lysed in ice-cold RIPA buffer containing 50 mmol/l Tris-HCl buffer (pH 7.6), 150 mmol/l NaCl, 1% Nonidet P40 Substitute, 0.5% sodium deoxycholate and 0.1% SDS. Protease inhibitor cocktail was purchased from Promega (Walldorf, Germany). Following a 30 min lysis period on ice, lysis samples were centrifuged at ∼20,000 g for 20 min at 4°C to prepare cell lysates. Then, 10 µg of lysate was subjected to SDS-PAGE on an 8-16% Tris-glycine gel (Invitrogen), followed by electroblotting onto an Immobilon PVDF membrane (EMD Millipore). ECL Western Blotting Substrate (07880, Nacalai Tesque) was used to detect signals. The following antibodies were used: monoclonal mouse anti-RREB1 (1:200; B-7, Santa Cruz Biotechnology), monoclonal rabbit anti-pSmad2/3 (1:1000; 8685, Cell Signaling Technology), monoclonal rabbit anti-Smad2/3 (1:1000; 8685, Cell Signaling Technology), monoclonal rabbit anti-pERK (1:1000; 4370, Cell Signaling Technology), polyclonal rabbit anti-ERK (1:1000; 9102, Cell Signaling Technology), α-tubulin (1:1000; T6074, Sigma-Aldrich), horseradish peroxidase-conjugated goat anti-rabbit IgG (1:500; 1706565, Bio-Rad) and horseradish peroxidase-conjugated goat anti-mouse IgG (1:500; 1706515, Bio-Rad).
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