Anti tublin

Immunoblot was carried out as described earlier (30 (link)). Briefly, the cells were lysed for 20 min on ice in RIPA lysis buffer containing a protease inhibitor cocktail and DMSF. The samples were subjected to 12% SDS-PAGE and transferred to nitrocellulose membranes. Blots were probed with primary antibodies anti-Caspase-3 (Abcom, 1:1,000), anti-Cyto c (Abclone, 1:1,000), anti-Tublin (Affinity, 2:1,000), and anti-β-actin (TransGen, 1:2,000) at 4°C overnight with gently shaking. After three times washing with PBS, the horseradish peroxidase (HRP)-conjugated secondary antibodies were added. Antigen-antibody complexes were visualized by enhanced chemiluminescence. Enhanced ECL TM prime detection reagent (30 (link)) was used to visualize antigen-antibody complexes, and the density was quantified by ImageJ.

The proteins were extracted through the radio-immunoprecipitation assay (RIPA) buffer that contains protease inhibitor cocktail (Sigma, St. Louis, MO, USA) and phenylmethane-sulfonyl fluoride (Sigma). Protein concentration was determined using the Pierce™ BCA Protein Assay Kit (Thermo Scientific). Proteins were separated by 8–12% Tris-acrylamide gels and transferred onto the Immobilon-P Transfer Membranes (Merck KGaA, Darmstadt, Germany). The membranes were incubated with primary antibodies at 4 °C overnight and secondary antibodies at room temperature for 1 h. The primary antibodies used were as follows: anti-Ki67 (1:1000; Affinity, Cincinnati, OH, USA), anti-PCNA (1:1000; Affinity), anti-TUBLIN (1:1000; Affinity), and anti-THOC1 (11,000; Abcam, Cambridge, UK). The proteins were visualized using the ECL reagent (Millipore) and photographed using an electrophoresis gel imaging system (ChemiScope 6000, CLIX, Shanghai, China).