The cell pellet was resuspended in 10 ml harvest buffer (Dulbecco's PBS, Sigma, plus 0.4 g/ml CsCl) and the cells lysed by sonication.
The cell pellet was washed with 5 mls of Harvest Buffer (20 mM Hepes pH 7.4, 2 mM MgOAc, 100 mM KOAc), pelleted again as before, resuspend in 1 mL of harvest buffer, transferred to an eppendorf, pelleted with brief (10 sec) spin, supernatant was aspirated off, and the pellet was lysed immediately (see below) or stored pellet at −20°C.
Cellular proteins were solubilized in ice-cold harvest buffer containing 0.5% Nonidet P-40, 50 mM Tris-HCl (pH 8.0), and 10 µM PMSF following the treatments indicated.
Lysed cells were resuspended in lysis buffer (harvest buffer brought up to high salt concentration (0.5 M KCl) and containing 1 mM PMSF).
Beads were then washed with TEV buffer (harvest buffer without protease inhibitors), and incubated overnight at 4°C with purified GST-TEV (tobacco etch virus) protease to cleave off Cdc24.
After 24 hr, cells were lysed in harvest buffer and subjected to GST affinity purification.
A standard curve was produced from bovine serum albumin (BSA) (Fisher, Pittsburgh, PA) diluted in harvest buffer.
Sample, diluted sample, or standard measuring 10 μL was added to 90 μL harvest buffer in a 96-well plate.
Intracellular staining for STAT proteins: PDCs were harvested using TEN harvest buffer and fixed with 4% formaldehyde by 10-min incubation at 37°.
After 48 h, cells were harvested at 4°C in cold harvest buffer and spun down at 4°C at 3000 r.p.m. for 5 min.
