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lonal isolates of infectious, low passage B. burgdorferi sensu stricto were utilised for all the experiments. B. burgdorferi was cultured in Barbour Stoenner Kelly medium at 37 C as previously described. Phagocytosis assays were performed as previously described. Briefly, coverslips in 24 nicely plates were coated with 1% rat collagen in 60% ethanol GDC-0152 remedy and dried overnight. Fully differentiated BMDMs were plated in RPMI supplemented with 30% L cell conditioned media, 20% FBS and 1% penicillin streptomycin. Cells were maintained in this media for 24 hours after which placed into serum free RPMI overnight prior to use in assays. Serum free circumstances were utilised for experimentation to provide uniformity in the media and to avoid cross reaction with bovine cytokines and inhibitors present in serum.
B. burgdorferi were added towards the cultures at a multiplicity of infection of 10. Plates were centrifuged at 1200 rpm at 4 C for 5 min to bring B. burgdorferi in get in touch with with all the cells. To initiate phagocytosis, the plates were moved to 37 C. Coverslips were removed at various timepoints soon after the addition of B. GDC-0152 burgdorferi and washed with cold PBS three times to get rid of unbound B. burgdorferi. Cells were fixed in 3. 7% paraformaldehyde with 5% sucrose in PBS for 20 min at 25 C. Coverslips were washed three times in phosphate buffered saline and stored at 4 C until use. For experiments with poly I:C stimulation, cells were treated with synthetic double stranded RNA for 4 hours before phagocytosis assay was performed. For experiments with interferon stimulation, macrophages were primed with either recombinant or IFN overnight prior to phagocytosis.
For experiments with pathway inhibitors, the inhibitors were added towards the cells 1 h prior to addition of B. burgdorferi. U0126, SP600125, AG490, RO31 8220 and LY294002 were purchased from Calbiochem. The concentrations of the inhibitors Siponimod utilised were in conformity with earlier published reports and had no visible cytotoxic effect on the BMDMs as judged by trypan blue exclusion. The activity of the inhibitors at the concentrations was confirmed by testing for known effects of the inhibitors on expression of selected genes by q rt PCR. Immunofluorescence microscopy Immunofluorescence studies were performed as previously described with all the following modifications. Briefly, the coverslips were incubated three times for 5 min in blocking buffer at room temperature.
All antibody incubations were continued for 1 h at 37 C in a humidified incubator. After blocking, the coverslips were incubated for 1 h at 37 C with an anti B. burgdorferi polyclonal rabbit antibody diluted 1:10, 000 in blocking buffer. Coverslips were then washed three times with blocking buffer and incubated with a fiTC conjugated goat anti rabbit IgG antibody. Samples were once more washed Messenger RNA three times in phosphate buffered saline for 5 min after which permeabilized with chilled methanol for 10 sec. After incubating three times for 5 min in blocking buffer, the coverslips were once more incubated with Siponimod the anti B. burgdorferi rabbit antibody. After washing three times for 5 min in blocking buffer, samples were incubated simultaneously with a Texas Red conjugated goat anti rabbit IgG antibody.
For studies of Arp3 localization, Arp2/3 complexes were detected by rabbit anti Arp3 antibody, a generous GDC-0152 gift of Dr. Ralph Isberg and B. burgdorferi were identified by using mouse anti OspA antibody. To determine the number of special BLAST hits we followed the method described in. To determine Siponimod members of signaling pathways as described by the KEGG database, we manually annotated the G. bimaculatus transcriptome as described in. Briefly, BLAST was utilised to evaluate the sequences of D. melanogaster pathway members with all the G. bimaculatus transcriptome assembly along with the best hit was selected as a putative ortholog with an E value cutoff of e 10. To ascertain whether the de novo assembly contained members of previously known G.
bimaculatus GenBank accessions, we utilised tBLASTn or BLASTn to query the G. bimaculatus transcriptome GDC-0152 assembly. For automatic annotation of all transcriptome sequences, we designed a custom script known as Gene Predictor. This script assigns putative gene orthology depending on comparisons with all the D. melanogaster proteome, downloaded as described in Table S1. A protein BLAST database was produced utilizing the D. melanogaster proteome. A nucleotide BLAST database was produced utilizing the non redundant assembly items of the G. bimaculatus de novo transcriptome assembly. The best 50 BLAST hits for every sequence of the D. melanogaster proteome compared with all the G. bimaculatus transcriptome were obtained Siponimod utilizing the TBLASTN algorithm and stored in a MySQL database. Reciprocally, the best BLAST hit for every sequence of the G. bimaculatus transcriptome against the D. melanogaster proteome was obtained utilizing the BLASTX algorithm and stored within a separate MySQL database. A custom script then iterates through every of the entries of the D. melanogaster prote