Anti inducible nitric oxide synthase

Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and other tissue culture reagents were purchased from Gibco BRL Co. (Grand Island, NY, USA). Lipopolysaccharide was obtained from Sigma-Aldrich (St. Louis, MO). Primary antibodies such as anti-inducible nitric oxide synthase (iNOS) and anti-cyclooxygenase-2 (COX-2) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-p-extracellular signal-regulated kinase (ERK), anti-ERK, anti-p-c-Jun N-terminal kinase (JNK), anti-JNK, anti-p-p38, and anti-p38 were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-mouse, anti-goat, and anti-rabbit secondary antibodies were supplied by Merck Millipore (Darmstadt, Germany). The enzyme-linked immunosorbent assay (ELISA) kit for PGE₂ was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Endothelial cells were seeded at the density of 50.000/well in 24 multiwell plates in medium with 10% serum. After cell adhesion, NPs or lyophilized biomaterials were dissolved in medium and placed in direct contact with the cells. After 24 h incubation, biomaterials were gently removed and cells were lysed as described in [20 (link)]. Cell lysates were centrifuged at 10000 g for 20 min at 4°C. Following gel electrophoresis, proteins were blotted onto activated nitrocellulose membranes, incubated overnight with the anti caspase-3 antibody (1:1000) (Cell Signaling, Danvers, USA), anti p21 antibody (1:1000, Cell Signaling), anti p53 antibody (1:1000) (Santa Cruz, Heidelberg, Germany), anti inducible nitric oxide synthase (iNOS, 1:1000, Santa Cruz) or anti cyclooxygenase-2 (COX-2) antibody (1:1000) (Cayman, Ann Arbor, USA). The primary antibody was detected by incubating the membranes for 1 h with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody (Promega, Madison, USA)) diluted 1:2500 in PBS, followed by enhanced chemiluminescence detection system (Bio-Rad, Hercules, USA). Images were digitalized with CHEMI DOC Quantity One. Results were normalized to those obtained by using an antibody anti β-actin (1:10000) (Sigma). Original blots are reported as Supplemental Information S1 Fig.

2,4-Dinitrophenol (DNP)-specific IgE (D8406), DNP-conjugated bovine serum albumin (DNP-BSA, #A6661) and compound 48/80 (#C2313) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies were purchased from commercial sources: anti-phospho-extracellular signal-regulated kinase (p-ERK, #4370) and anti-phospho-p38 mitogen-activated protein kinase (p-p38^MAPK, #9211) (Cell signaling, Beverly, MA, USA); anti-actin (Sigma-Aldrich, #A2066); anti-cyclooxygenase-2 (COX-2, #sc-1747), anti-inducible nitric oxide synthase (iNOS, #sc-7271) and anti-IgG horseradish peroxidase conjugates (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).