Rabbit anti p21 antibody

The western blot analyses were performed as described in our previous work (19 (link), 23 (link)). The primary antibodies were as follows: rabbit anti-SirT1 and rabbit anti-AC-P53 (Lys373, Lys382) antibodies (Millipore, Billerica, MA, USA); rabbit anti-P-Akt (Ser473), rabbit anti-Akt, rabbit anti-P-GSK-3β (Ser9), rabbit anti-GSK-3β, rabbit anti-cleaved-caspase-3, mouse anti-P53 and rabbit anti-β-Actin antibodies (Cell Signaling Technology, Beverly, MA, USA); mouse anti-Bax and mouse anti-Bcl-2 antibodies (BD Biosciences, San Diego, CA, USA) and rabbit anti-P21 antibody (Abcam, Cambridge, UK). After washing, proteins were detected using horseradish peroxidase (HRP)-conjugated anti-rabbit and HRP-conjugated anti-mouse secondary antibodies (Cell Signaling Technology, Beverly, MA, USA). The optical density (OD) values of the immunoblot bands were quantified with Scion Image analysis software (Scion Corp., Frederick, MD, USA). The OD values were normalized using appropriate internal controls (optical density detected protein/optical density internal control).

Mononuclear cells from the controls and primary AML patients were washed twice with phosphate buffer saline and resuspended in 200 μl of lysis buffer containing a mix of protease inhibitors (Beyotime, Haimen, Jiangsu, China). Immunoblots were prepared as previously described [22 (link)]. Rabbit anti-GDF15 monoclonal antibody (Abcam, Cambridge, MA, USA), rabbit anti-β-tubulin antibody (Abcam, Cambridge, MA, USA), rabbit anti-Cdk2 antibody (Abcam, Cambridge, MA, USA), rabbit anti-Cyclin D1 antibody (Abcam, Cambridge, MA, USA), rabbit anti-P21 antibody (Abcam, Cambridge, MA, USA) and rabbit anti-β-actin monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA). The THP-1 cell CM was obtained from cells cultured in regular medium with 1% FBS at different cell densities (2 × 10⁵/ml, 5 × 10⁵/ml, 1 × 10⁶/ml, 2 × 10⁶/ml). The ELISA analysis for GDF15 was performed according to the manufacturer’s instructions (R&D systems).

The protein was extracted from the gastric tissue using an extraction buffer. The protein concentrations were determined by the bicinchoninic acid assay (BCA) method using the BCA Protein Assay Kit (Beyotime Institute of Biotechnology, China). Total protein extracts were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels. After electrophoresis, the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). After blocked in tris-buffered saline (containing 5% milk and 0.3% Tween 20 (TBST)) for 1 h, membranes were probed with monoclonal antibodies, rabbit anti-p21 antibody (Abcam, Cambrige, UK), and rabbit anti-mammalian target of rapamycin (mTOR) antibody (Affinity Biosciences, Cincinnati, USA). After incubation overnight, the membranes were washed with TBST and incubated with Goat anti-rabbit IgG (H + L) horseradish peroxidase (HRP; Affinity Biosciences, Cincinnati, USA). The blots were visualized by the SuperSignal enhanced chemiluminescence (ECL) detection system according to the manufacturer’s instructions (Bioshine ChemiQ 4600 fluorescence and chemiluminescence imaging system, Ouxiang Scientific Instrument Co. ltd, Shanghai, China). The relative band intensity was then quantified.