Goetz, University of Tuebingen, Germany). S. pneumoniae, strain TIGR4 Δcps is the non-encapsulated variant of TIGR4 (provided by S. Hammerschmidt, University of Greifswald, Germany). Bacteria were cultured on Columbia sheep red blood agar plates (bioMérieux, Nuertingen, MG-132 nmr Germany) and incubated at 37°C overnight. Monocytes were stimulated with bacteria at a ratio of 1:5 cells/bacteria. IRAK4, MyD88, and the respective control Stealth RNAiTM and LipofectamineTM RNAiMAX reagent were obtained from Invitrogen (Karlsruhe, Germany). siRNA-mediated gene knockdown experiments were performed in a 96-well format,
adapted from the Invitrogen reverse transfection protocol. Real-time RT-PCR was conducted using the High Pure RNA Isolation Kit (Roche, Mannheim, Germany), Superscript III First strand cDNA synthesis https://www.selleckchem.com/products/AZD6244.html kit (Invitrogen, Karlsruhe, Germany), Absolute QPCR SYBR GREEN Low ROX Mix (ABgene House, Epsom, UK), and a 7900 HT Fast Real Time PCR System (Applied Biosystems, Darmstadt, Germany) following the manufacturers´ instructions. Relative expression was calculated by normalization to β-actin mRNA expression levels as rE =
1/(2Ct(target) − Ct(beta-actin)). Primers were obtained from MWG Biotech (Ebersberg, Germany): Human β-actin (F 5′-AGAGCTACGAGCTGCCTGAC-3′; R 5′-AGCACTGTGTTGGCGTACAG-3′; 184 bp); irak4 (F 5′-GCCACCT-GACTCCTCAAGTC-3′; R 5′-CAAATCCTCCCTCTCCCATT-3′; 115 bp); myd88 (F 5′-GACTGCTCGAGCTGCTTACC-3′; R 5′-GCGGTCAGACACACACAACT-3′; 193 bp); il-10 (F 5′-ACGGCGCTGTCATCGATT-3′; R 5′-GGCATTCTTCACCTGCTCCA-3′; 167 bp); socs3 (F 5′-GCCACTCCCTGGGAGTCC-3′; R 5′-ATAGGAGTCCAGGTGGCCGT-3′; 151 bp); socs1 (F 5′-CCTGGTGCGCGACAGC-3′; R 5′-CAGCAGCTCGAAGAGGCAGT-3′; 138 bp); tnfr2 (F 5′-TGAAAAAGAAGCCCTTGTGC-3′; R 5′-CTGTGGCTGGTTCCGAGT-3′; 188 bp); foxo3 (F 5′-GGGGAACTTCACTGGTGCTA-3′; R 5′-GAGAGCAGATTTGGCAAAGG-3′; 143 bp), foxo1 (F 5′-AGGCTGAGGGTTAGTGAGCA-3′; R 5′-GCCAAGTCTGACGAAAGGAA-3′; 170 bp). Supernatants from monocytes were collected after
24 h. Cytokine levels (TNF, IL-10, IL-12p40, IL-6, and IL-1β) were quantified using the BDOptEIATM kits selleck products (BD Biosciences, Heidelberg, Germany). For the NF-κB ELISA, nuclear extracts were prepared 30 min after LPS stimulation with a nuclear extract kit (Active Motif, La Hulpe, Belgium). Transcription factor activity was quantified with the TransAM NF-κB Transcription Factor Assay Kit (Active Motif). For protein lysates cells were harvested by centrifugation and washed in PBS. The pellet was resuspended in RIPA lysis buffer containing aprotinin, leupeptin, PMSF, NaF, and Na3VO4 (all from Sigma). After incubation on ice for 30 min lysates were centrifuged at 13 000 rpm for 15 min at 4°C, supernatants collected and stored at ‒20°C. 12% SDS-PAGE was performed with equal amounts of whole cell lysates of 1–2×106 monocytes and protein transfer to nitrocellulose membrane (Whatman, Dassel, Germany) by semi-dry blotting.