Ibot dry blotting system

Transferrin receptor and RAGE expression level in human cells was determined by Western blot analysis. Briefly, cells were seeded in 75 cm²-flasks and grown to reach 60–70% confluence. The cells were washed in ice-cold PBS three times and then scraped off the flask and collected by centrifugation. The pelleted cells were lysed for 10 min at room temperature on a shaker (900 rpm) following the ProteoJET™ Mammalian Cell Lysis Reagent protocol. Lysates were spun in a centrifuge at 18,000 × g for 15 min and the supernatant was collected. Equal protein aliquots were resolved by SDS-PAGE, transferred to nitrocellulose membranes using iBot Dry Blotting system (Invitrogen, CA, USA), immunoblotted with primary antibodies (mouse anti-human TfR antibody or rabbit polyclonal to mouse and human RAGE) (1;200 and 1:750, respectively) and detected with HRP-Goat-anti-mouse (or rabbit) IgG (H + L) secondary antibody (1:3000). Tubulin was immunoblotted with mouse anti-human-β-tubulin (1:200) and detected with HRP-Goat-anti-mouse IgG (H + L) secondary antibody (1:3000). These were followed by incubation with Novex® ECL Chemiluminescent Substrate Reagent Kit. The Bands were quantified with ImageJ 1.44p (http://imagej.nih.gov/ij/).

Proteins with an equal amount (10 μg from each sample) were resolved by 12% SDS-PAGE at 150 V for approximately 2 h using a SE260 Mini-vertical Electrophoresis Unit (GE Healthcare). The resolved proteins were then transferred onto a PVDF membrane using the iBot dry blotting system (Invitrogen; Carlsbad, CA). Nonspecific bindings were blocked with 5% (w/v) skim milk in PBS at RT for 1 h. The membrane was incubated with mouse monoclonal anti-EF-Tu (Hycult biotech; Uden, The Netherlands) (1:5,000 in 1% (w/v) skim milk/PBS) at 4 °C overnight. After washing, the membrane was further incubated with rabbit anti-mouse IgG conjugated with horseradish peroxidase (Southernbiotech; Birmingham, AL) (1:10,00αn 1% (w/v) skim milk/PBS) at RT for 1 h. For loading control, the membrane was incubated with goat polyclonal anti-GAPDH antibody conjugated with horseradish peroxidase (Abcam; Cambridge, UK) (1:300 in 1% (w/v) skim milk/PBS) at 4 °C overnight. Immunoreactive protein bands were developed with Amersham ECL Prime Western blot detection reagent (GE Healthcare) and visualized with ImageQuant Las 4000 (GE Healthcare). Band intensity was measured by ImageQuant TL software (GE Healthcare).

After DEP treatment as described above, protein was extracted from collected SAEC using RIPA buffer (Sigma-Aldrich, Cat. No. R0278) containing protease inhibitor cocktail set III (Calbiochem, Cat. No. 539134).
Protein was separated by electrophoresis with a Nupage gel and transferred to a PVDF membrane with the iBot Dry blotting system (Invitrogen). For Western blot analysis, proteins of interest were probed with specific primary antibodies against Nrf2 (Santa Cruz, Cat. No. sc-722), pAMPK (Cell Signaling, Cat. No. 2532 s), and β actin (Abcam, Cat. No. 8224) as an internal control, at appropriate dilution from 1000–5000. Secondary antibodies were horseradish peroxidase conjugated (HRP) goat anti-rabbit IgG (Abcam, Cat. No. ab6721) and HRP goat anti-mouse IgG (Abcam, Cat. No. ab6789), at a 5000–20,000 of dilution. The signals were detected with Amersham ECL Plus (GE Healthcare, Cat. No. RPN2132). Each treatment sample was repeated with at least three biological replicates. ImageJ (1.47 V) was used to quantify the band intensities by subtracting background signal of each band (underneath area). The real signal values of Nrf2 and p-AMPK in all groups were normalized by the intensity of β actin bands. The comparisons among each group were performed by one-way ANOVA using Origin 2018.