Antibodiesdetecting cleaved parp and caspase 3

Total protein concentration was measured by bicinchoninic acid assay kit
(Sigma Aldrich, Shanghai, China). The same quality of protein (15 μg)
was fractionated on 4–15% polyacrylamide gels and transferred onto
nitrocellulose (Bio-Rad, Philadelphia, PA, USA). The levels of MTDH
were analyzed by western blot using an anti-MTDH at 1:700 dilution
(Thermo Fisher Scientific, Shanghai, China). Additionally, antibodies
detecting cleaved PARP and caspase-3 (1:1000; Cell Signaling, Beverly,
MA) were used to assess apoptosis activity. Membranes were washed
three times with TBST and incubated with the goat anti-rabbit
horseradish peroxidase-conjugated secondary antibody (1:2000; Bio-Rad,
Philadelphia, PA, USA). Normalization was performed by blotting the
same samples with an antibody against β-actin (Abcam, Burlingame, CA,
USA).

Total protein lysates were fractionated on 4%-15% polyacrylamide gels and transferred onto nitrocellulose (Bio-Rad). These primary antibodies were used: anti-MTDH at 1:500 dilution (2F11C3 monoclonal antibody, Thermo Fisher), anti–phospho-p65 at 1:1000 with anti–total p65 at 1:1000 (Cell Signaling Technologies), anti–c-Myc at 1:500 (Abcam), and anti–β-Actin (Sigma-Aldrich). Additionally, antibodies detecting cleaved PARP and caspase-3 (Cell Signaling) were used to assess apoptosis activity. Subsets of samples were randomly selected from the original group of 41 tumors and 14 normal pleurae for this series of experiments. All protein experiments were performed in triplicate. To avoid problems related to incomplete membrane stripping, separate blots were used for each independent experiment.