Imaging station

To measure the oligomycin resistant phenotype of yeast expressing various substrates, wild-type or doa10Δ yeast containing a vector or the Yor1-GFP, Yor1-GFP-Ub₄, Pca1(1-392)-Yor1-GFP, and Pca1(1-392)-Yor1-GFP-Ub₄ were grown overnight to stationary phase. Six 5-fold dilutions were made and transferred to YPEG solid medium supplemented with 2.5 μg/ml oligomycin (dissolved in DMSO). The plates were incubated at 26°C for 5 days and imaged 5 using a Bio-Rad imaging station.

For experiments that examine whole cell lysates, BMMs were cultured with M-CSF and RANKL for various periods of time and then processed for western analysis. For all western blot analysis, equal amounts of lysates were loaded and electrophoresed on sodium dodecyl sulfate–polyacrylamide electrophoresis gels using a 10% running gel under reducing conditions. Separated proteins were transferred to nitrocellulose membranes, and the membranes were probed with the indicated antibodies, which except for the antibody to PAR1 from LifeSpan Biosciences (Seattle, WA), were all from Cell Signaling Technology (Danvers, MA).
Reactive bands were detected by enhanced chemiluminescence using Chemiluminescence LumiGLO (CellSignaling Technology). Detection was by autoradiography. Quantification was achieved by digitalizing the blots using an imaging station (BioRad, Hercules, CA) and then measuring band density
To detect p65 and phospho-p65, BMMs (5×10⁵cells/well in 6-well plates) were treated with M-CSF + RANKL (30 ng/ml M-CSF and 60 ng/ml RANKL) for 3 days to assure induction of PAR1, transferred to serum free medium for 3 hours and then treated with RANKL (60 ng/ml) for 15 minutes. Cells were lysed in cold lysis buffer and separated into cytoplasmic and nuclear fractions using a commercial kit (Cell Signaling Technology).

Mouse colons and mouse embryonic fibroblasts (MEFs) were used as the source of the material for immune-blotting (western blotting). Animals were euthanized, colons extracted and pulverized by sonication in a commercially available animal tissue extraction buffer following the protocols in (Kaneda et al., 2007 (link)). The cells were cultured following the protocols from the same study. Briefly, cells were serum starved overnight, treated to experimental conditions and harvested in denaturing SDS based lysis buffers, suspended in 4x loading buffer and heated to 95 degC for 5 minutes and stored for archival at −20degC. Lysates (about 20μg) were reduced with DTT and subjected to gelelectrophoresis in precast Bis-Tris gels under reduced conditions and transferred to 0.2μm Nitrocellulose membrane in a dry-transfer iBlot apparatus. Membranes were blocked with 5%BSA in PBS and then incubated with the relevant primary antibody followed by the secondary with 5 to 6 washes in washing buffer in between. The membranes were developed using PicoWest chemiluminescence substrate and imaged using a BioRad imaging station. The images of the blots were then quantified using the gel band intensity analysis macro in ImageJ. Several independent experiments were performed, as indicated, to assure biological reproducibility and statistical power of analysis.