Tbs blotto a

Proteins were extracted from OS tissues or cells using Tissue Extracts & Cell Lysates (Santa Cruz, San Diego, CA, USA), and 50 µg proteins were applied to carry out the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 90 min. After proteins were transferred onto the UltraCruz Nitrocellulose Pure Transfer Membranes (Santa Cruz), TBS Blotto A (Santa Cruz) was exploited as a blocking reagent to prevent the binding of no-specific protein-binding signals. Then the membranes were incubated with primary antibodies in the diluted solution at 4°C overnight, followed by the combination of secondary antibody and primary antibodies at room temperature for 1 h to form the protein complex. The objective protein levels were analyzed by detecting the intensity of the immunoconjugated signals through Western Blotting Luminol Reagent (Santa Cruz) under the ImageLab software version 4.1 (Bio-Rad, Hercules, CA, USA). All the antibodies were bought from Abcam (Cambridge, UK): anti-Beclin-1 (ab62557, 1:1000), anti-light chain 3B (anti-LC3B; ab51520, 1:1000), anti-P62 (ab109012, 1:1000), anti-HDAC4 (ab12172, 1:1000), internal control anti-GAPDH (ab9485, 1:3000) and secondary antibody goat anti-rabbit IgG/HRP (ab205718, 1:5000).