Tumor cell lysates (TCLs) have been reported to induce antitumor immunity; however, it remains unclear which elements serve a role in this process. The present study identified 768 proteins that were upregulated in TCL prepared from Lewis lung cancer cells compared with the lysate from type II alveolar epithelial cells. Among the proteins that were upregulated in TCL, receptor for activated C kinase 1 (RACK1) and catenin β‑like 1 (CTNNBL1) are closely associated with cell proliferation and the inhibition of apoptosis. To determine the role of these proteins in TCL, a protein extraction method was designed, which was based on immunoprecipitation. Using this method, RACK1 and CTNNBL1 were extracted, whereas the other proteins within the TCL were not affected. The modified TCL exhibited a stronger ability to induce splenocyte apoptosis, whereas the ability to promote cell activation was reduced. These findings suggested that the TCL depends on RACK1 and CTNNBL1 to activate mouse immunocytes, including monocytes and B lymphocytes, and inhibit apoptosis. Therefore, the present study may provide information regarding the composition of TCLs and their positive regulatory effect on immunocytes.