Ab205270

OE tissues were lysed in lysis buffer for 30 min on ice. The lysis buffer consisted of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% Triton X-100, 5 mM sodium fluoride, 1 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 2 mM sodium orthovanadate, and a protease inhibitor cocktail (P8340, Sigma). The lysates were then centrifuged at 12,000 rpm for 30 min to obtain the suspended proteins. The proteins were extracted after adding 5× loading buffer and denatured at 100°C for 10 min before being separated by SDS-PAGE (8%–12%). The proteins were then transferred into nitrocellulose membranes (Life Sciences) and immersed in 5% skim milk for blocking at room temperature for 1 h, followed by an overnight incubation with primary antibodies at 4°C, then rinsed with Tris-buffered saline with Tween 20 and incubated for 1 h at room temperature after adding the appropriate secondary antibodies (1:5,000, Bio-Rad). Primary antibodies included anti-p-YAP (Ser127, 1:1,000, #4911, CST), anti-YAP (1:1,000, ab205270, Abcam), anti-TAZ (1:1,000, DF4653, Affinity), anti-MOB1 (1:1,000, #13730, CST), and anti-p-MOB1 (Thr35, 1:1,000, #8699, CST). Anti-GAPDH (1:5,000, #2118, CST) and anti-β-actin (1:10,000, A5316, Sigma) were used as loading controls. Protein bands were detected through ECL (Bio-Rad), and analyzed by Quantity One software (Bio-Rad).