Supplementary Methods
Centrifugal Elutriation Exponentially growing asynchronous cell cultures were
separated in a Beckman JE-6B elutriating rotor system. Cells were elutriated in
growth medium at room temperature at a constant flow rate of 40 ml/min as described
previously (Zachos et al., 2005). The different cell cycle populations were obtained
by slowing the rotor to various predetermined speeds. Flow cytometry analysis of
DNA content, bromodeoxyuridine (BrdU) incorporation, and phospho-serine 10
histone H3 positive cells was used to accurately quantify the proportion of G1, S and
G2/ M phase cells in each fraction.
Cell Cycle Analysis Cells were fixed in 70% ethanol in PBS overnight. For DNA
content analysis cell were pelleted and resuspended in PBS containing 1 g/ml RNase
(Qiagen) and 10 g/ml propidium iodide, incubated at room temperature for 30 mins,
and then analyzed using a Beckton Dickinson FACScan flow cytometer. To monitor
BrdU incorporation, cells were incubated with 20 M BrdU for 30 min, fixed and
incubated with anti-BrdU antibody followed by FITC-conjugated secondary antibody
as described previously (Zachos et al., 2003). For mitotic index determinations fixed
cells were incubated with anti-pS10 histone H3 primary antibody followed by FITC
conjugated secondary antibody as described (Zachos et al., 2005). Anti-BrdU or antipS10 histone H3 stained cells were counterstained with propidium iodide as above
and analyzed for FITC and PI fluorescence as above.
Cell Lysis DT40 cells were lysed in ice cold lysis buffer (LB, 20 mM Tris acetate,
pH 7.5, 0.27 M sucrose, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 10 mM
sodium ß-glycerophosphate, 5 mM sodium pyrophosphate, 1 mM sodium
orthovanadate, 50 mM sodium fluoride, 0.1% 2-mercaptoethanol, 10 mg/ml leupeptin,
20 mg/ml aprotonin, 1.2 mM benzamidine, 10 mg/ml soybean trypsin inhibitor, 2
mg/ml pepstatin). Lysates were snap frozen, thawed on ice and cleared by
centrifugation at 16,000g for 15 min.
Immunoprecipitations Chk1 was immunopreciptated from 0.25-1 mg of cell lysates
prepared as described above using 1 g of the indicated antibodies, following preclearing with 1 g rabbit IgG (Sigma). Antibody conjugates on protein G-sepharose
beads (Sigma) were washed either three times in LB (native conditions) or three times
in RIPA buffer (stringent conditions) as indicated in the text and once in PBS. For
western blotting the precipitates were re-suspended in 2 x laemmli sample buffer and
boiled for 5 min before being run out on 10% SDS-PAGE gels, transferred to
nitrocellulose membrane and blotted with the appropriate antibodies.