Antibody for β actin

Mc-fEE (also called Nonitri®) and β-glucan were provided by COSMAXNBT Inc. (Korea). RAW264.7 cells (ATCC number TIB-71) were purchased from the American Type Culture Collection (ATCC) (Rockville, MD, USA) and YAC-1 (KCLB number 40160) cells were purchased from the Korean Cell Line Bank (Seoul, Korea). Roswell Park Memorial Institute (RPMI) 1640 media, antibiotics (penicillin–streptomycin solution), and phosphate buffered saline (PBS) were purchased from Hyclone (Logan, UT, USA). Stain buffer (FBS) and specific antibodies [FITC CD3, PE CD4, PE-Cy^TM7 CD8, FITC CD11c, PE CD335 (NKp46), PE-Cy ^TM7 F4/80] were purchased from BD Biosciences (Sparks, MD, USA). NK Cell Isolation Kit (mouse), buffer (0.5% BSA, 2 mM EDTA in PBS, pH 7.2), and autoMACS® Rinsing Solution (2 mM EDTA in PBS, pH 7.2) were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Antibodies for p65, p50, c-Jun, c-Fos and their phosphor-specific antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody for β-actin was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). TNF-α ELISA kit (Quantikine™ ELISA, Cat No.: MTA00B) was purchased from R&D Systems (Minneapolis, MN, USA).

Total proteins (50-100 μg) extracted from cell lines or human NSCLC and adjacent normal tissues were analyzed by SDS-polyacrylamid gel electrophoresis and were transferred electrophoretically to nitrocellulose membrane. To evaluate expression of the proteins, blots were blocked with 5% nonfat milk in Tris-Buffered Saline and Tween 20, and incubated with a primary rabbit monoclonal antibody Pim-1, Cyclin D1, CDK4 or eIF4E. Antibody for β-actin (Santa Cruz Biotechnology, Santa Cruz, CA) was used as a control. The blots were then re-probed with secondary antibody and visualized by the chemiluminescence and scanned using ImageQuant LAS 4010 Imaging System (GE Healthcare Life Sciences, Piscataway, NJ).