Anti cdk6

Total protein of the leukocytes of CML patients and CML IR cell lines were extracted and isolated through SDS–polyacrylamide gel electrophoresis (Beyotime), and transferred onto a polyvinylidene fluoride membrane (Millipore, Burlington, MA, USA). The following primary antibodies were used for the assay: anti-GS (Affinity, Jiangsu, China), anti-mTOR (Affinity), anti-Cyclin D1 (Affinity), anti-CDK4 (Affinity), anti-CDK6 (Affinity), anti-PCNA (Affinity), anti-Tubulin (Proteintech, Rosemont, IL, USA), anti-GAPDH (Proteintech), and anti-β-actin (Proteintech).

First, the spleen paraffin sections were deparaffinized in xylene and graded ethanol. To block endogenous peroxidase activity, the sections were incubated with 10% hydrogen peroxide. For antigen retrieval, the sections were heated in 2% EDTA solution for 15 min, and allowed to cool for 2 h at room temperature. After washing with PBS, the slides were blocked with goat serum (Zhongshanjinqiao Biotechnology Co., Ltd., China) for 15 min, and then incubated with the diluted antibodies at 4℃ overnight. On the next day, the sections were incubated with corresponding secondary antibodies at room temperature for 15 min. The sections were then labeled with horseradish peroxidase, and the signals were detected using the DAB Peroxidase Substrate Kit (Solarbio). After staining with hematoxylin (Beyotime) for 30 s, the slides were dehydrated in graded ethanol and xylene. Finally, the sections were sealed with coverslips by resinene (Solarbio). The following primary antibodies were used for the assay: anti-GS (Affinity), anti-mTOR (Affinity), anti-Cyclin D1 (Affinity), anti-CDK4 (Affinity), anti-CDK6 (Affinity).

Total proteins were extracted using RIPA lysis buffer supplemented with protease inhibitor Cocktail (Roche, Switzerland). Protein concentrations were calculated by using BCA protein assay kit (Beyotime, China). Equivalent amounts of protein samples (50 μg) of each group were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a PVDF membrane. Membranes were blocked in 5% nonfat dried milk and incubated with the primary antibodies overnight at 4°C. After several washes, membranes were incubated for 1 h at room temperature with corresponding second antibody and detected by enhanced chemiluminescence (ECL) assay kit (Millipore, USA). The primary antibodies were as follows: anti-p21 (1/2000) (Cell Signaling Technology, USA), anti-Cyclin D1 (1/2000) (Affinity, USA), anti-CDK4 (1/1000) (Affinity, USA), anti-CDK6 (1/2000) (Affinity, USA), anti-Cyclin A2 (1/400) (Boster, China), anti-GAPDH (1/500) (Boster, China), and anti-α-tublin (1/500) (Boster, China).