Anti cyclin d1

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).

As described previously, we extracted proteins from cells with RIPA lysis buffer, separated equal amounts of protein using SDS-PAGE, and then transferred the protein to a 0.45-µm PVDF membrane (Millipore, USA). Next, the membrane was blocked with 5% skim milk for 1 h at room temperature before it was washed 3 times with 1× TBST for 10 min each time. The membranes were then incubated with the following antibodies: rabbit anti-GAPDH (#5174), anti-Notch2 (#5732), anti-Bax (#0120), anti-Bcl-2 (#6139), anti-γH2AX (#8482), anti-Cyclin D1 (#0931), anti-AKT (#6261), anti-phospho-AKT (#0016), anti-mTOR (#6308), and anti-phospho-mTOR (#3308), all of which were purchased from Affinity Biosciences (OH, USA). A chemiluminescence reagent was used to visualize the protein bands, and the grayscale values of the bands were measured with Image Lab 6.0 software (Bio-Rad) [19 (link)].

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).