For Western blots cells were grown to 80%90%90% subconfluent monolayers and processed as described elsewhere [ 23] using a monoclonal antibody against Smad4 (clone B-8, Santa Cruz Biotechnology Inc, Santa Cruz, California, USA), or a monoclonal antibody against α-tubulin (Zymed Laboratories Inc ,San Francisco, California, USA).
For western blotting cells were grown in 25 cm flasks and exposed to drugs as specified.
For Western blot, cells were grown as indicated above in the co-culture system.
For CCS (copper chaperone to superoxide dismutase) Western blot, cells were grown on six-well plates, transfected and treated with the specified compounds.
For western blots, RAW cells were grown in six-well dishes at 100,000 cells/well with the same cytokine treatments.
For Western blot analysis, cells were grown in 6-well plates, washed with PBS, harvested with a rubber policeman, and collected by brief centrifugation.
In brief, for the quantitative western blot assays, cells were grown on dishes and then scraped into 1x Laemmli (4% SDS, 5% 2-mercaptoethanol, 10% glycerol, 0.002% bromophenol blue, 0.0625 M Tris-HCl; pH 6.8).
P19 Cells and Western Blotting Mouse embryonal carcinoma P19 cells were grown in DMEM supplemented with 10% FCS, 2 mM glutamin and 1% penicillin/streptomycin (all from GIBCO) and transfected with lipofectamin according to the manufacturer's protocol (Lipofectamin® 2000 reagent, Invitrogen).
Immune Blot Analysis To monitor Hsp90 protein levels, cells were grown overnight in YPD at 30°C with or without TSA (25 μg/mL for S. cerevisiae, 6 μg/mL for C. albicans ) as indicated.
Following transfection, cells were grown for 24 48 h for Western blot analysis or further processed for cyst culture or proliferation assay.
Cells were grown in normal media for 24 h before transfection, and were used for Western blot and IF 48 h after transfection.
