Enhanced chemiluminescence system

Sub-confluent A549 were seeded in 6 cm diameter Petri dishes. After adherence, cells were treated for the indicated times with Ni(SalPipNONO) (0.5 mM) and specific pathway inhibitors. Protein extraction and Western blot were performed as previously described [58 (link), 61 (link)]. Electrophoresis (50 μg of protein/sample) was carried out in 4–12% Bis-Tris Gels (Life Technologies, Carlsbad, CA, USA). Proteins were then blotted onto nitrocellulose membranes, incubated overnight with primary antibodies [Anti-cytochrome c, anti-phospho-ERK1/2, anti-caspase- 3, anti-ERK antibodies from Cell Signaling (Celbio, Milan, Italy); anti-p53 antibody from Santa Cruz Biotechnology Inc (Dallas TX, USA); anti-COX-2 from Cayman Chemical (Ann Arbor, MI, USA); anti-HIF-1α from BD Biosciences (San Jose, CA, USA); anti-VEGF from Merck KGaA (Darmstadt, Germany)] and then detected by enhanced chemiluminescence system (Biorad, Hercules, CA, USA). Results were normalized to those obtained by using an antibody against beta actin from Merck KGaA (Darmstadt, Germany) or total ERK1/2 (Cell Signaling, Celbio, Milan, Italy), when indicated.

Western blotting process was performed as described previously [23 (link)]. In brief, each LD sample was homogenized in ice-cold RIPA buffer with PMSF protease inhibitor buffer (Beyotime, Shanghai, China) and centrifuged (12,000 × g) for 30 min at 4 °C. Then, the supernatant of each sample was collected and the total protein concentration was measured using the BCA kit (no. A045-3, Nanjing Jiancheng Bioengineering Institute, China). Protein samples (20 μg) were separated by using 8%−12% SDS-PAGE gels and transferred onto polyvinylidene difluoride membranes (Bio-Rad, CA, USA). The above membranes were blocked with 5% defatted milk for 2 h and washed three times with TBST (prepared with Tris–HCl buffer and isotonic salt solution and contains 1% Tween 20) and incubated overnight at 4 °C with primary antibodies (detailed in Additional file 1: Table S4). Then, these membranes were incubated with corresponding secondary antibodies (anti-rabbit or anti-mouse IgG, 1:5,000, Proteintech Group, IL, USA). The bands were visualized by using an enhanced chemiluminescence system (Bio-Rad, CA, USA), and the densitometric of Western blot bands were analyzed using the Image Lab™ software system (Bio-Rad, Hercules, CA, USA).

Three control air-porch mice and fifteen air-porch bone graft mice were used for western blot analysis. Three control air-porch mice without PE induction were used as the control group (1), and fifteen air-porch bone graft mice were randomly divided into five groups (n = 3 in each group): (2) saline; (3) SCNPs; (4) SCNPs + MF; (5) ZSCNPs; (6) ZSCNPs + MF. To maximize the therapeutic effect of NPs, 200 µl of 10 mg/ml NPs or saline was intravenously injected into the mice every 48 h, and a magnet (maximum MF strength = 24.5 Gs) was placed on the air-porch surface for 48 h in groups 4 and 6. After 14 days, the whole air-porch samples were collected and the proteins in air-porch wall tissues were extracted for further assays. The collected proteins were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes, and blocked with bovine serum albumin in Tris-buffered saline containing 0.05% Tween 20. Rabbit anti-c-Fos, rabbit anti-NFATc1, rabbit anti-IκBα, mouse anti-p-IκBα and mouse anti-β-tubulin primary antibodies were incubated with membranes at 4°C overnight, followed by incubation with fluorescent secondary antibodies for 2 h at room temperature. An enhanced chemiluminescence system (Bio-Rad Laboratories, Hercules, CA) was utilized to measure the levels of specific proteins after different treatments.