Twist 25465 1 ap

Experimental cells were harvested and then mixed with RIPA:PMSF (100:1) to facilitate total protein extraction, and protein concentration was then determined using a bicinchoninic acid kit (BOSTER). Equal concentrations (40 μg) of total protein were then separated by electrophoresis on a 10% separation gel and transferred to a polyvinylidene fluoride membrane. This membrane was then blocked for 1 h using nonfat milk (5%) and incubated overnight with a working solution of the relevant primary antibodies (EFEMP2 12004-1-AP, STEAP1 20199-1-AP, STEAP2 20201-1-AP, Twist 25465-1-AP, Proteintech; STEAP3 PA5-115969, STEAP4 PA5-115971, Invitrogen™; EMT Antibody Sampler Kit #9782, Cell Signaling Technology; PI3K ab86714, p-PI3K ab182651, AKT ab8805, p-AKT 38449, mTOR ab32028, p-mTOR ab109268, Abcam) at 4°C. The membranes were then washed using Tris-buffered saline with Tween® 20 and incubated with the secondary antibody for 1 h at room temperature. Membranes were then washed again and visualized using an enhanced chemiluminescent substrate kit (Millipore) and Image J software.

Western blotting was performed using an enhanced chemiluminescence kit (Merck Millipore), as described previously.¹⁵ The following antibodies were used: myc (PL14) and β‐actin (M177‐3) from MBL (Nagoya, Japan); E‐cadherin (#3195), N‐cadherin (#13116), Smad2/3 (#8685), phospho‐Smad2 (#3108), phospho‐Smad3 (#9520), Snail (#3879), Slug (#9585), HMGA2 (#5269) from Cell Signaling Technology (Beverly, MA); TEAD4 (ab58310) from Abcam (Cambridge, UK); GFP (mFX75) from Fujifilm Wako (Tokyo, Japan); TetR (TET01) from MoBiTech (Cambridge, MA, USA); and TWIST (25465–1‐AP) from Proteintech (Rosemount, IL, USA). Horseradish peroxidase (HRP)‐F(ab’)2 secondary antibodies were purchased from GE Healthcare (Waukesha, WI, USA). Protein bands were analysed using a ChemiDoc XRS+image analyzer (Bio‐Rad, Hercules, CA, USA). The intensity of bands was measured by Quantity One software (Bio‐Rad). Quantitative ratios of E‐cadherin, N‐cadherin, Snail, Slug, HMGA2 or TEAD4 to actin and EGFP to TetR, which were calculated based on the data, are shown as relative values.