Proteome profiler mouse array panel a kit

Tumor samples were mechanically and chemically disaggregated with radioimmunoprecipitation assay buffer and sonicated on ice. 20–50 µg of total protein were loaded on SDS‐PAGE gels and transferred to polyvinylidene difluoride membranes (Invitrogen, Thermo Fisher Scientific). Membranes were incubated overnight at 4°C with the primary antibodies biotin anti-CD45 (clone 30F11, Affymetrix), andhorseradish peroxidase (HRP)-conjugated anti-TATA-box binding protein (TBP). Membranes were washed with 0.1% PBS‐Tween 20 and stained using anti-CD45 with DAB solution (SK-4100, Vector Laboratories) for 15 min. Proteins were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Burlington, Massachusetts, USA) and images were acquired using ChemiDoc MP Imaging System (Biorad).
Extracted proteins from the tumor samples were also used to study the pro-inflammatory cytokines in the tumor microenvironment. Three hundred micrograms of protein from a pool of three tumors per group were used to perform the cytokine array assay following manufacturer’s indications (Proteome Profiler Mouse Array Panel A kit, ARY006, R&D Systems, Minnesota, USA). Cytokine expression was measured semi-quantitatively by pixel density of duplicated spots using ImageJ software.