X omat ar autoradiography film

Protein was extracted from tumor nodules collected from control-untreated and treated tumors using RIPA buffer (50 mM Tris–HCL, pH 8.0, with 150 mM sodium chloride, 1.0% Igepal CA-630 (NP-40), 0.5% sodium deoxychlorate, and 0.1% sodium dodecyl sulfate) with protease inhibitor. The lysate from normal lung tissues was also prepared in a similar manner as described above. Protein content was measured using BCA Protein Assay Reagent Kit (PIERCE, Rockford, IL). Equal amounts of supernatant protein (50 µg) from the control and different treatment groups were denatured by boiling for 5 min in sample buffer, separated by 10% SDS-PAGE, transferred to nitrocellulose membranes for immunoblotting. Membranes were blocked with 5% skim milk in Tris-buffered saline with Tween 20 and probed with antibodies against ER stress markers and β-actin (1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA). Horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were used. Proteins were visualized using enhanced chemiluminescent solution (Pierce, Rockford, IL) and exposed to Kodak X-OMAT AR autoradiography film (Eastman Kodak, Rochester, NY).

The protein extraction, sample preparation, SDS page and transfer of proteins to nitrocellulose membrane were performed according to our previous methods [8 (link),9 (link)]. Membranes were probed with antibodies against cleaved caspase 3 (1:1000), Cyclin D1 (1:1000), PCNA (1:1000), and β-actin, and all these primary antibodies were purchased from Santa Cruz Biotechnology, Santa Cruz, CA. Horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were used. Proteins were visualized using enhanced chemiluminescent solution (Pierce, Rockford, IL) and exposed to Kodak X-OMAT AR autoradiography film (Eastman Kodak, Rochester, NY). The intensity of the western blot bands was quantified by densitometric analysis using imageJ software.