To prepare for experiments using washed solids, whole slurry PCS was washed using a basket centrifuge (Model STM-2000, WeStatesStates, Hamilton, OH, USA) with a nylon filter bag (105 μm pore size, 25% open area, Sefar filtration cloth, Western States) until the glucose concentration in the wash water was below 0.1 g/l.
Inoculums were freshly prepared prior to experiments using frozen stocks of S. aureus (ATCC strain 29213, frozen at midlog phase; optical density 625 = 0.8).
Conditioned media were retained for hormone assays, and at the end of culture either viable cell number was determined (dose response experiment) by neutral red uptake assay [ 17] or cell lysates were prepared (microarray experiment) using the lysis buffer component of the RiboPure RNA isolation kit (Ambion/ Life Technologies Ltd., Paisley, UK).
CLL-BC nuclear extracts were prepared and pull-down experiments using biotin-labeled STAT3-DNA probes were performed.
FRAP experiments (using cells prepared as above) were instead performed on a Zeiss LSM 710 M laser scanning microscope using a 63 × Plan-Apochromat NA 1.4 oil objective.
Representative Western blotting results are shown in Fig. 4a, and data from three independent experiments using individually prepared lysates are summarized in Fig. 5a.
We have repeated these experiments using chromatin prepared from pupae because it is during this stage that the adult cuticle is elaborated.
Stuart Mossman, a visiting clinical fellow, undertook the first passive transfer experiments, using IgG prepared from plasma exchange samples and demonstrating a defect in nerve-evoked muscle responses in the injected mice with little effect on AChR numbers (Supplementary material B79).
However, we could not detect RUNX2/p53 complex by co-immunoprecipitation experiments using cell lysates prepared from untreated cells.
Co-immunoprecipitation (Co-IP) experiments using cell lysates prepared from S2 cells co-expressing HA-tagged Arl1 and FLAG-tagged Arfip incubated with anti-FLAG antibody coated beads finds that the anti-FLAG coated beads can successfully co-precipitate both the Arfip-FLAG target and Arl1-HA (arrowhead; Fig. 1E, lane 3).
