Supplementary information.
Materials and methods
Immunoblot analysis and quantification of band intensity
Whole cell lysates were prepared in RIPA buffer supplemented with 5L/mL Protease
Inhibitor Cocktail III (Calbiochem), 0.5mM phenylmethylsulfonyl fluoride and 1mM
NaVO4 (Sigma-Aldrich). For isolation of nuclear protein the cells were lysed in 500 L
buffer A (20mM HEPES [pH 7.9], 10mM NaCl, 3mM MgCl2, 0.5% Noindet P-40, 10%
glycerol, 0.2mM EDTA, Protease Inhibitor Cocktail III [Calbiochem]). After a 20-minute
incubation on ice, nuclei were pelleted by centrifugation. Pellets were washed once more
with 300 L buffer A, and repelleted before the addition of 300 L of buffer B (20mM
HEPES, [pH 7.9], 20% glycerol, 0.2mM EDTA, Protease Inhibitor Cocktail III). Nuclei
were resuspended in buffer C (20mM HEPES [pH7.9]. 0.4 M NaCl, 20% glycerol,
0.2mM EDTA, Protease Inhibitor Cocktail III) incubated on ice, and periodically
vortexed for 1 hour. Nuclear extracts were then collected after a 15 min centrifugation.
The proteins were quantified using the Bradford assay, and loaded onto 8-12.5% SDS
polyacrylamide gels. Gel semi-dry transfer to a polyvinylindene difluoride membrane
(Millipore) was performed. The blots were probed with primary antibodies overnight at
4C, incubated with horseradish peroxidase-conjugated secondary antibody for 1 hour at
RT, and detected by an enhanced chemiluminescence system (Amersham Biosciences).
The immunoblots were quantified using Image Quant Mac v1.2 software. Background
values were subtracted from quantified protein levels. The c-Myc protein levels were
normalized to actin protein levels, and c-Myc protein turnover was graphed as percent of
c-Myc remaining after cycloheximide treatment using Excel, with 100 percent of protein
at time zero.
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Immunoprecipitation
BC-3 cells were resuspended in lysis buffer (50 mM Tris pH 7.5, 50 mM NaCl, 0.2%
Nonidet P-40, 2 % glycerol, 0.5 mM EDTA, 1mM dithiothreitol, 0.5mM
phenylmethylsulfonyl fluoride [PMSF] and protease inhibitor cocktail) at 4°C for 15 min.
The lysate was cleared by centrifugation. The proteins were quantified by the Bradford
method and 10 mg of total whole cell lysate was precipitated with rabbit anti-GSK-3 at
a dilution of 1:50 or control rabbit antibody overnight. The precipitate was washed six
times with buffer, resuspended in 20 L loading buffer, separated by SDS–
polyacrylamide gel electrophoresis and probed with mouse monoclonal anti-c-Myc, rat
monoclonal anti-LANA or mouse anti-GSK-3 antibodies.
RNA extraction and quantitative RT-PCR
RNA was extracted using Trizol reagent (Invitrogen). DNase-treated total RNA (1.0 g)
was reverse transcribed with the reverse transcription system (Promega) and resuspended
in 100 L of sterile distilled water. The primers used were c-Myc forward, 5’-AAG GCC
CCC AAG GTA GTT ATC C-3’ and reverse, 5’-TTT CCG CAA CAA GTC CTC TTC
A-3’; and -actin forward, 5’-CCTGGCACCCAGCACAA-3’ and reverse, 5’GCCGATCCACACGGAGTACT-3’.
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