Ab83009

Beads-homogenized tissue or MECs (EasySep, STEMCELL Technologies) were lysed in ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer (with phosphatase and protease inhibitors) and sonicated for 5 min at high or low intensity, respectively (Standard Bioruptor Diagenode). Cellular debris was pelleted (15,700g, 15 min, 4°C), and protein concentration was determined by the DC Protein Assay (Bio-Rad). Equal amounts of proteins were separated by SDS–polyacrylamide gel electrophoresis. After transfer onto nitrocellulose membranes (Sigma-Aldrich, GE10600001), membranes were blocked for 1 hour in 5% milk, and specific proteins were labeled with the corresponding primary antibodies against vinculin (Abcam, ab18058), CPEB3 (Abcam, ab10883), CPEB2¹⁹, CPEB4 (Abcam, ab83009), CPEB1 (Cell Signaling Technology, no. 13583), CyclinD1 (Santa Cruz Biotechnology, sc-717), CREB1 (Cell Signaling Technology, no. 9197), α-tubulin (Sigma-Aldrich, T9026), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Life Technologies, AM-4300). Secondary HRP antibodies were also diluted in 5% milk, and proteins were revealed using enhanced chemiluminescence Western blotting detection reagents (GE Healthcare).