Opcml

Total protein of cells was extracted by lysis buffer and concentration determination was conducted using the DC protein assay method of Bradford (Bio-Rad, Hercules, CA). Twenty mg of protein was loaded and separated in 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to polyvinylidine difluoride membranes (Millipore, Bedford, MA). The antibodies used for the analysis are as follows, OPCML (Abcam, Cambridge, UK), cyclin-dependent kinase inhibitor 1B (p27), cleaved caspase 3, cleaved caspase 9, poly ADP-ribose polymerase (PARP), phospho-Protein Kinase B (AKT) (Ser473) and phosphor-glycogen synthesis kinase 3β (GSK3β) (Cell Signaling Technology, Inc., Danvers, MA), and GAPDH (Good Here Biotechnology, Hangzhou, P.R. China).

The second aliquot of the lung tumor samples was used to determine protein expression of the TSGs. The TSGs showing significant promoter demethylation after aerosol Aza treatment as analyzed by the methylation qPCR array were selected for evaluation by western blot. The frozen tissues were homogenized in cold RIPA buffer with protease inhibitor cocktail (Sigma) using a hand homogenizer, followed by sonication. Protein samples were prepared for Western blot following a routine protocol from Life technologies (Grand Island, NY). Primary antibodies against human H-cadherin, OPCML, Rassf1a (Abcam), SFRP1 (Epitomics, Burlingame, CA), and beta-actin (Santa Cruz Biotechnology) were used to identify the targeted proteins. LI-COR C-DiGit Blot Scanner was used to scan the western blots; the bands were analyzed by Image Studio software (LI-COR). The density ratio of targeted protein vs. actin loading control of each sample was used to present the protein expression levels semi-quantitatively.