Louis, MO)

Louis, MO). sepsis, and inhibition of PKD1 activation together with antibiotic treatment in GBS-infected neonates could be an effective way to control GBS diseases. and assay. The sequences of S-ODN used have been previously reported (41). Peptidoglycan (PGN) was purchased from Invivogen (San Diego, CA). PMA, ionomycin, and D-galactosamine (D-GalN) were purchased from Sigma Chemical Co. (St. Louis, MO). G?6976, G?6983, and CRT0066101 were purchased from Tocris (Minneapolis, MN). IRAK inhibitor 1 (IRAK4 inhibitor; 6-imidazo[1,2-a]pyridin-3-yl-N-piperidin-4-ylpyridin-2-amine) was purchased from APExBIO (Houston, TX). In vivo experimental protocol To investigate role of PKD on antibiotic-killed GBS-mediated proinflammatory responses, C57BL/6 mice were injected intraperitoneally (i.p.) with vehicle (7.6% AMG-3969 v/v DMSO in PBS), PKC inhibitor G?6983 (2.3 mg/kg) or a PKD inhibitor G?6976 (2.3 mg/kg) 4 h and 1 h before the antibiotic-killed GBS (2 108 cfu/mouse) challenge. Two hr later, blood and spleen samples were obtained to prepare serum, cell extracts, and total RNA. To investigate role of TLR signaling modulators on GBS induced shock-mediated death of D-GalN-sensitized mice, mice (C57BL/6, MyD88?/?, or TLR9?/?) were challenged with the antibiotic-killed GBS (2 108 cfu/mouse) plus D-GalN (30 mg) by i.p. injection. In some experiments, C57BL/6 mice were injected i.p. with vehicle, G?6983 or G?6976 4 h and 1 h before and 2 h after the antibiotic-killed GBS plus D-GalN challenge. Fifteen mg of penicillin G was injected daily for the first 3 days to ensure complete killing of GBS. Viability of mice was observed up to 8 days. Preparation of whole cell lysates and Western blot analysis Whole cell lysates were prepared from RAW264.7 cells or whole spleen cells as previously described (42). To detect the presence or phosphorylation status of specific proteins in whole cell extracts, equal amounts of whole cell lysates were subjected to electrophoresis on a 10% polyacrylamide gel containing 0.1% SDS, and then Western blots were performed using specific antibodies, as previously described (42). All phospho-specific Abs were purchased from Cell Signaling (Beverly, MA). Antibodies specific for actin, PKD, IB or IB were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). In vitro kinase assays FLAG-tagged PKD-expressing AMG-3969 RAW264.7 cells were stimulated with GBS. Each FLAG-tagged PKD protein in whole cell lysates was immunoprecipitated with anti-FLAG Ab. The resulting immune complexes were subjected to kinase assay using Syntide-2 (Sigma) as a PKD substrate, as previously described (43). Cytokine-specific enzyme-linked immunosorbent assay (ELISA) Levels of selected cytokines in culture supernatant or serum were analyzed by cytokine specific ELISA as described AMG-3969 previously (44). All recombinant murine (rm) cytokines, antibodies specific for murine cytokines and recombinant human cytokines were purchased from BD Biosciences (San Diego, CA). Preparation of DNA-free RNA and RT-PCR DNA-free total ATN1 RNA was isolated from RAW264.7 cells or spleens by using the RNeasy Mini Kit (Qiagen Inc., Valencia, CA) following the manufacturers protocol. To measure the relative amount of selected gene transcripts, isolated RNA (1 g from each sample) were reverse transcribed with oligo(dT) primer using Superscript II reverse transcriptase (Moloney murine leukemia virus reverse transcriptase; Invitrogen). One tenth of the cDNA product was then amplified with gene specific primers. Twenty to forty cycles of PCR were conducted. PCR products were separated by 1% agarose gel electrophoresis and visualized. The sequences of RT-PCR primers for mouse genes are previously described (38, 45). The sequences of RT-PCR primers for human genes are: TNF (F:5 CCTGTAGCCCATGTTGTAGC3, R:5CAAAGTAGACCTGCCCAGAC3), IL-6 (F:5ACTCACCTCTTCAGAACGAA3, R:5CTCAAACTCCAAAAGACCAG3), IL-8 (F:5CACCCCAAATTTATCAAAGA3, R:5TCAAAAACTTCTCCACAACC3), and -actin (F:5GTGGGCGCCCCAGGCACCA3, R:5CTCCTTAATGTCACGCACGATTTC3). All primers were purchased from Integrated DNA Technologies, Inc. (Coralville, IA). Flow cytometric analysis To analyze cell surface expression of CD86, cells were stained with APC-conjugated rat antiCmouse CD86 or APC-conjugated isotype control. CD86 expression was analyzed with BD FACSAria II flow cytometer (BD Biosciences, San Diego, CA) and FlowJo flow cytometry data analysis software (FlowJo LLC, Ashland, OR). All Abs were purchased from BD Biosciences. Statistical analysis All experiments were repeated at least three times before analysis. Data are expressed as the mean S.D. of triplicates. Two-tailed Students < 0.05, < 0.005, and < 0.001 are indicated as *, **, and #, respectively, and considered significant. RESULTS Live GBS and antibiotic-killed GBS induce activation of PKD1 We previously found that.