Can get signal 1

3 x 10⁵ WT and Ddx6 KO ESCs were plated into the well of a 12-well plate with feeder cells in 2i-LIF medium. Two days later, cells were treated with 10 ng/ml rBMP4, 0.6 μM LDN193189, or 10 mM SB431542 (FUJIFILM/Wako) for 3 hrs, and 25 ng/ml of ActivinA for 1 or 2 hrs at 37°C. ESCs were harvested with TNE buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, and 1% NP40) supplemented with cOmplete Protease inhibitor cocktail (Roche), and centrifuged. Next, the supernatant concentration was adjusted, mixed with 3XSDS buffer (0.2M Tris-HCl pH6.8, 9% SDS, 30% glycerol, 15% 2-mercaptoethanol, 0.006% bromophenol blue), and incubated at 96°C for 5 min. After SDS-PAGE, proteins were transferred onto a Immobilon-P Transfer Membrane (Millipore). The membrane was blocked with 3% skim milk /TBS-T, and then incubated with primary antibodies diluted in Can Get Signal 1 (TOYOBO, NKB101) for phospho-Smad1/5 or phospho-Smad2 antibodies or 3% skim milk /TBS-T for other antibodies at 4°C overnight and subsequently incubated with a secondary antibody at room temperature for 90 min. Detection was performed using the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) and images were acquired with a ChemiDoc Touch MP system (Bio-Rad).

Cultured tissue cells were lysed in lysis buffer supplemented with a protease inhibitor cocktail and disrupted on ice using a sonicator. The protein concentration of cell lysates was assessed using the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA) with bovine serum albumin as a standard. Each cell lysate (20 µg) was subjected to SDS-PAGE using a 5-20% gel (Wako, Osaka, Japan) and transferred to a PVDF membrane. After blocking with PVDF-blocking reagent (Toyobo, Osaka, Japan), 1 μg/mL of purified polyclonal antibody for each antigen in Can Get Signal 1 (Toyobo) were incubated with the membrane. Immunoreactive antigens were detected using anti-rabbit IgG HRP-linked antibody (Cell Signaling Technology, Tokyo, Japan) and Western Lightning Plus ECL (PerkinElmer, Waltham, MA, USA). The control monoclonal antibodies to MAGE-A4 (clone: E701U, Cell Signaling Technology, Tokyo, Japan) and XAGE-1b (clone: USO9-13) (47 (link)) were employed for the validation of the specificity of polyclonal antibodies. The membrane was reprobed with an anti-β-tubulin antibody (Wako).