Goat antimouse immunoglobulin

Isolation of proteins from MNCs of CLL was conducted as previously reported [43 (link)] and 30 μg of proteins were tracked on gels and transferred to polyvinylidene difluoride membranes. After this process, the membranes were blocked in 4% milk solution (Serva Electrophoresis GmbH, Heidelberg, Germany) for 1 h at +40 °C. Then, the membranes were incubated with appropriate primary antibodies. The primary antibodies used in our study were against S100A4 (Cell Signaling Technology, Inc.), S100A8 (Abcam, Cambridge, UK), S100A9 (Abcam), S100A12 (Elabscience Biotechnology Co., Ltd, Wuhan, China), β-actin (R&D Systems, Inc, Minneapolis, MN, USA), phospho-NF-κB p65 (Ser536, Cell Signaling Technology), and NF-κB (Santa Cruz Biotechnology, Dallas, TX, USA). Goat antirabbit immunoglobulin (R&D Systems) was used as a secondary antibody, except for β-actin where goat antimouse immunoglobulin was used. The degree of expression of the tested proteins was determined by densitometric analysis and the ChemiDoc Imaging System (Bio-Rad Laboratories, Hercules, CA, USA). The obtained values were normalized to β-actin.

Proteins from MPN-derived MNC were isolated and processed as previously reported [9 (link)]. Equal amounts of protein (30 μg) were run on polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 4% milk (Serva Electrophoresis GmbH, Heidelberg, Germany) for 1 h at room temperature and probed with primary antibodies directed against HIF-1α (Elabscience, Wuhan, China), VEGF (Elabscience), eNOS (Elabscience), β-actin (R&D Systems, Inc, Minneapolis, Minnesota), phospho-STAT5 (R&D Systems), STAT5 (R&D Systems), phospho-AKT (R&D Systems), AKT (R&D Systems), pmTOR (Cell Signaling Technology Inc., Beverly, USA) and mTOR (Cell Signalling Technology). Peroxidase-conjugated goat anti-rabbit immunoglobulin (R&D Systems) was used as a secondary antibody, except goat anti-mouse immunoglobulin (R&D Systems) was used for β-actin. The protein levels were imaged with a ChemiDoc Imaging System (Bio-Rad Laboratories, Hercules, CA, USA) and estimated by densitometric scanning of the blots using the Image Lab (Bio-Rad Laboratories, Inc. Version 6.0.0.25) software tool and normalized to β-actin.