Anti ptbp1

Proteins were extracted from cells by radio immunoprecipitation assay (RIPA) buffer and quantified using bicinchoninic acid assay. Equal protein amounts were loaded on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and followed by transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% skimmed milk, membranes were incubated with anti-PTBP1 (1:10,000; Abcam) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:10,000; Abcam) at 4 ℃ overnight, followed by goat anti-rabbit IgG (1:5,000; Abcam) at room temperature for 1 h. Protein bands were visualized by the enhanced chemiluminescence kit.

Cells and tissues were lysed in a radio immunoprecipitation assay (RIPA) lysis buffer supplemented with protease inhibitors (Roche). Total protein extracts were separated by 8% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred to a polyvinylidenefluoride (PVDF) membrane (Millipore). The membranes were blocked in 5% skimmed milk for 1 hr and incubated with anti‐PTBP1 (1:1,000; Abcam), anti‐PTEN (1:1,000; Abcam), anti‐Akt (1:1,000; Abcam), anti‐p‐Akt (1:1,000; Abcam), anti‐LC3B (1:1,000; Abcam), anti‐Flag (1:2,000; ShangHai Ruixing), anti‐Actin (1:5,000; Abcam, as a loading control), and anti‐glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (1:15,000; KangChen, China, as a loading control) antibodies. Appropriate primary antibodies were added at 4°C overnight. Appropriate secondary antimouse and antirabbit antibodies were added for 2 hr at room temperature. Finally, for detection of the proteins, an enhanced chemiluminescence (ECL) chemiluminescence system (Thermo Fisher Scientific/Thermo pierce) was used.