Total ikba

Sample proteins were resolved by SDS-PAGE and transferred onto Immobilon-P membranes (Millipore Corp., Bedford, MA, USA). The primary antibodies phosphorylated-Akt (Ser473), total Akt, total IkBa (Cell Signaling), and Sirt1 (Santa Cruz), were used. Protein was detected by horseradish peroxidase-conjugated secondary antibody (Bio-Rad, Hercules, CA, USA) and visualized by a SuperSignal West Pico Chemiluminescent Substrate Kit (Pierce, Rockford, IL, USA).

BMDMs generated as aforementioned were seeded onto 6-well plates at a density of 2 × 105 cells/well and serum starved overnight. Cell-free DNA was secreted by AE17, or AE17 cells treated with karonudib (10 μM, overnight) isolated from culture supernatants using a commercial kit (Nucleospin, Macherey-Nagel Düren, Germany). BMDMs were subsequently treated with 20 ng/mL extracellular DNA for 2 h. TLR9 inhibitor chloroquine (Merck, Darmstadt, Germany) was used at 2 μg/mL for 40 min before addition of cfDNAs. Cell lysates were prepared and analyzed by Western blotting for phospho-p65 NFKB (#3031), total p65 NFKB (#4764), phospho-ikBa (#2859) and total-ikBa (#4812) (Cell Signaling Technology Inc., Danvers, MA, USA). Results were normalized to actin (#4970, Cell Signaling Technology Inc., Danvers, MA, USA). Visualized bands were quantified by GelPro Analyzer 6.3 (Media Cybernetics, L.P., Rockville, MD, USA)

PBMC were cultured with anti-CD3 and anti-CD28 in the presence or absence of RDE for 72 h. Mice splenocytes were cultured with the Th17 condition in the presence or absence of RDE for 72 h. Both cells were then harvested and lysed with lysis buffer. Protein concentration was measured using the Bradford method (Bio-Rad, Herculed, CA, USA). Protein samples were separated using 12% SDS-PAGE and transferred onto nitrocellulose membranes (AmersharmPharmacia Biotech, Piscataway, NJ, USA). For Western blot hybridization, the membrane was preincubated with blocking buffer for 2 h and then incubated with primary antibodies against Total IkBa, p-IkBa, Total ERK, p-ERK, Total STAT5, pSTAT5, Total STAT3, pSTAT3(727), pSTAT3(705) (all from Cell Signaling, Danvers, ma), and β-actin for 1 h. After washing, horseradish peroxidase-conjugated secondary antibodies were added, and the membranes were incubated for 1 h at room temperature. After washing, the hybridized bands were detected using an ECL detection kit (Pierce, Rockford, IL, USA) and Hyperfilm (Agfa, Belgium).