Pure nitrocellulose membrane

The cells were washed and homogenized by protein RIPA lysis buffer as described previously [36 (link),37 (link)]. Soluble proteins were collected after centrifugation at 13,200 rpm for 15 min at 4°C and protein content was determined using the BCA protein assay reagent kit. Aliquots of the extract containing 30 μg of protein were loaded and run on a single track of 10% SDS–PAGE and then transferred onto a pure nitrocellulose membrane (Bio-Rad, Hercules, CA, U.S.A.). The blots were blocked with 5% non-fat milk and then incubated overnight at 4°C with primary antibodies: mouse monoclonal anti-TfR1 (1:1000), rabbit polyclonal anti-DMT1 (1:1000), rabbit polyclonal anti-Fpn1 (1:1000), rabbit polyclonal anti-Ft-L (1:1000), mouse monoclonal anti-IRP1 (1:1000), anti-IRP-2 (1:1000) overnight at 4°C. After being washed three times, the blots were incubated with goat anti-rabbit (1:5000) or anti-mouse IRDye800 CW secondary antibody (1:5000) for 2 h at room temperature. The intensities of the specific bands were detected and analyzed by the Odyssey infrared imaging system (Li-Cor, Lincoln, NE, U.S.A.). To ensure even loading of the samples, the same membrane was probed with a mouse monoclonal anti-β-actin antibody (1:5000) as internal protein controls.

In brief, cells growing in 100 mm dishes were rinsed twice with cold PBS and then lysed on ice for 20 min in 1 ml lysis buffer (40 mM Hepes pH 7.5, 120 mM NaCl, 1 mM EDTA, 10 mM pyrophosphate, 10 mM glycerophosphate, 50 mM NaF, 0.5 mM orthovanadate, and EDTA-free protease inhibitors (Roche)) containing 1% Triton X-100. After centrifugation at 13,000Xg for 10 min, samples containing 20-50 μg of protein were resolved by SDS-PAGE and proteins transferred to Pure Nitrocellulose Membrane (Bio-Rad Lab.), blocked in 5% nonfat milk, and blotted with the indicated antibodies. For immunoprecipitation experiments, the lysis buffer contained 0.3% CHAPS instead of 1% Triton. 4 μg of the indicated antibodies were added to the cleared cellular lysates and incubated with rotation for 6-16 hours. Then 25 μl of protein G agorose was added and the incubation continued for 1 h. Immunoprecipitates captured with protein G-agorose were washed three times with the CHAPS Lysis Buffer, two times by wash buffer A (50 mM Hepes, PH 7.5, 150mM NaCl), and boiled in 4x SDS samples buffer for western blot.

Culture cells were lysed in lysis buffer (Roche, IN) and centrifuged at 12,000g for 10 min to collect soluble proteins. Tissues were homogenized on ice in 300µl of 50mM Tris.Cl buffer (pH 7.0), followed by centrifugation at 12,000g for 10 min to collect supernatants. For Western blot, proteins (30 ~ 50 µg) were separated on 12% SDS-PAGE and blotted onto a pure nitrocellulose membrane (Bio-Rad, CA) at 180 mA for 2 hours. After being blocked with 5% skim milk in PBS at room temperature for 45 min, membranes were incubated with AKR1B10 (1:500) or acrolein (1: 500, Advanced Targeting System, CA) antibodies at 4^oC overnight, followed by incubation with goat anti-rabbit or anti-mouse IgG (1:2000; Sigma, MO) for 1 hour. Antibody binding signals were detected using enhanced chemiluminescence system (Pierce, IL). Protein loading amount was normalized by re-probing the membrane with β-actin monoclonal antibody (1:40,000; Sigma, MO).

Western blot was performed as described previously [10 (link)]. Briefly, cell lysates (30–50 μg) were subjected to SDS-polyacrylamide gel electrophoresis (SDS PAGE) and then subsequently transferred to pure nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA. Blots were visualized with a chemiluminescence ECL detection system (EMD Millipore, Rockford, IL, USA) and analyzed using a FujiFilm LAS-3000 imager (Fujifilm, Tokyo, Japan). HRP-conjugated sheep anti-mouse, donkey anti-rabbit secondary antibodies were from GE Healthcare UK Limited (Pittsburgh, PA, USA). PXDN antibody was from Abnova (Walnut, CA, USA). Rabbit polyclonal phospho-p53 and Bax antibodies were from Cell Signaling Technology (Danvers, MA, USA), while mouse monoclonal total p53 was from Santa Cruz Biotechnology, Santa Cruz, CA, USA. Mouse monoclonal β-actin antibody was from Sigma-Aldrich (St. Louis, MO, USA).

The cells were washed and lysed as described previously [47 (link),48 (link)]. After centrifugation at 13,200× g for 15 min at 4 °C, the supernatant was collected, and protein content was determined using the BCA protein assay kit. Aliquots of the extract containing about 20 µg of protein were loaded and run on a single track of 10% SDS-PAGE under reducing conditions and subsequently transferred to a pure nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The blots were blocked and then incubated with primary antibodies—rabbit monoclonal anti-phospho-JAK2 (1:1000), rabbit monoclonal anti-JAK2 (1:1000), rabbit polyclonal anti-phospho-STAT3 (1:1000), mouse monoclonal anti-STAT3 (1:1000), rabbit monoclonal anti-phospho-P65 (1:1000), rabbit monoclonal anti-P65 (1:1000), and mouse monoclonal anti-IRP1 (1:1000) antibodies—overnight at 4 °C. After the incubation, the blots were washed three times and then incubated with goat anti-rabbit (1:1000) or anti-mouse IRDye 800 CW secondary antibodies (1:5000) for 2 h at room temperature. The intensity of the specific bands was detected and analyzed by the Odyssey infrared image system (Li-Cor, Lincoln, NE, USA). To ensure even loading of the samples, the same membrane was probed with a mouse monoclonal anti-β-actin antibody at a 1:5000 dilution.