Anti phosphorylated erk1 2

Total protein was extracted from AgNP- and RA-exposed cells, as previously described [11 (link)], and the protein concentration of the supernatant was quantified using Bradford protein assay reagent (Bio-Rad Laboratories). Lysates (40 µg protein) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were then transferred onto nitrocellulose membranes. The membranes were then blocked using 5% (w/v) skimmed milk dissolved in Tris-buffered saline (TBS) with 1% (v/v) Tween-20 (TBST) for 1 h; incubated overnight with anti-phosphorylated AKT, anti-AKT, anti-phosphorylated ERK1/2, anti-β-Actin (all Santa Cruz Biotechnology, Dallas, TX, USA), and anti-ERK1/2 (Cell Signaling Technology, Beverly, MA, USA) primary antibodies, and then incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) for 2 h. All of the antibodies were diluted at the manufacturers’ recommended concentrations in 5% (w/v) dry milk dissolved in PBST. Protein signals were detected on X-ray films using an enhanced chemiluminescence kit (Amersham Biosciences, Piscataway, NJ, USA). The densitometry analysis was carried out using ImageJ software and the graphic data represent the ratio between the intensity of the proteins and the intensity of the housekeeping protein, β-Actin.

Recombinant mouse granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin (IL)-4 were purchased from JW CreaGene (Daegu, Korea). Lipopolysaccharide (LPS, from Escherichia coli O111:B4) were purchased from Invivogen (San Diego, CA, USA). The annexin V/propidium iodide (PI) kit was purchased from BD Bioscience (San Diego, CA, USA). ELISA kits for TNF-α, IL-6, IL-1β, IL-4, IL-12p70, and IL-10 were purchased from eBioscience (San Diego, CA, USA). The OT-I (OVA_257−264) and OT-II (OVA_323−339) peptides were synthesized by Abfrontier (Seoul, Korea). Fluorescein-conjugated antibodies (Abs) to, MHC-I, MHC-II, T-bet, and GATA-3 were purchased from eBioscience. Fluorescein-conjugated Abs to, CD11c, CD80, CD86, TNF-α, IL-12p70, IL-10, CD4, IFN-γ, IL-4, and CD8α were purchased from BD Bioscience. Anti-phosphorylated ERK1/2, -phosphorylated JNK, -phosphorylated p38, -phosphorylated IκB-a, IκB-a, NF-κB (p65), and lamin B were purchased from Santa Cruz Biotechnology (CA, USA). Anti-β-actin mAb (AC-15) and FITC-dextran (molecular mass, 40 kDa) were purchased from Sigma (St. Louis, MO, USA). All the pharmacological inhibitors were purchased from Calbiochem (San Diego, CA, USA).