binding to PR70, and that loss of PR70 causes increased levels of
Cdc6 and G
expressed sequence tag data base using the MegaBLAST tool
(www.ncbi.nlm.nih.gov/BLAST/) with the assembled PR70
sequence (21). A PR70 cDNA was constructed using the PR48
cDNA and the IMAGE Human Clone ID 5728169 (GenBank
using an internal NcoI restriction site present in the common
region of BM54432 and PR48. A PCR fragment containing the
translational start codon, the 5
with the PCR primers: 5
restriction enzymes BamHI and NcoI. The 3
and HindIII, and the fragments were ligated and subcloned into
the pCMV-Tag2B vector (Stratagene) digested with BamHI
and HindIII. The resulting construct encoded a full-length
PR70 cDNA fused to an N-terminal FLAG epitope tag. The
sequence was verified by automated sequencing.
modified Eagle’s medium containing 10% fetal bovine serum in
an atmosphere of 5% CO
transient expression of proteins, cells were transfected with
expression plasmids using Lipofectamine 2000 (Invitrogen)
according to the manufacturer’s protocol. Cells were harvested
either 24 or 48 h after transfection. Transfection with small
interfering RNA to knock down PR70 was carried out using
Oligofectamine (Invitrogen) following the manufacturer’s pro-
tocol. Annealed duplex siRNAs were purchased from Dharma-
con and had the following sequences: PR70-1, 5
protein kinase (22), an siRNA that knocks down protein phos-
phatase 5 (23), and an siRNA corresponding to the sequence of
firefly luciferase (5
transfection. In some experiments, cells were co-transfected
with PR70 siRNA and expression plasmids encoding wild-type
Cdc6 or Cdc6 mutants in which all three N-terminal phospho-
rylation sites were mutated to alanine (AAA-Cdc6) or aspartic
acid (DDD-Cdc6) using Lipofectamine 2000 and harvested 48 h
later. The cDNAs encoding phosphorylation site mutants of
Cdc6 were prepared using a PCR-based site-directed mutagen-
esis kit (Invitrogen) according to the manufacturer’s protocol.
Wild-type and mutant Cdc6 were expressed as a fusion proteins
fused to the N terminus of enhanced green fluorescent protein
using the pEGFP-N expression vector (Clontech).
terminus of human PR70 (CDLYEYACGDEDLEPL) conju-
gated to keyhole limpet hemocyanin. Anti-PR70 antibodies
were affinity purified on a peptide column made with the same
peptide using the MicroLink Peptide Coupling Kit (Pierce) fol-
lowing the manufacturer’s protocol. Rabbit antiserum against
human Cdc6 was generated against a full-length Cdc6 fusion
protein as described previously (4).
the cells were washed with cold PBS. The cells were incubated
on ice for 20 min in 300
the supernatant. Endogenous PR70 and Cdc6 were immuno-
precipitated from 1.2
a rabbit antiserum generated against the peptide CDLYEY-
ACGDEDLEPL conjugated to hemocyanin. Cdc6 was immuno-
precipitated using a rabbit polyclonal antibody generated
against a full-length Cdc6-GST fusion protein described previ-
ously. As a negative control, immunoprecipitations were per-
formed using pre-immune serum collected from the rabbits
immunized against PR70 or Cdc6. 10
protein-A beads were washed three times with IP lysis buffer,
and protein was solubilized in 60
on a 10% SDS-PAGE gel and transferred to a nitrocellulose
membrane. The membrane was cut into pieces, which were
probed with anti-PP2A C-subunit monoclonal antibody 1F6
(24), anti-PP2A A-subunit antiserum (C-20, Santa Cruz Bio-
technology), anti-Cdc6 monoclonal antibody (clone DCS-180,
Upstate), or anti-PR70 antiserum. Following incubation with
horseradish peroxidase-conjugated secondary antibodies, the
blots were developed using the enhanced chemiluminescence
detection system (Amersham Biosciences).
three times with lysis buffer and solubilized in 60
lulose membrane. The membrane was probed with anti-FLAG
M2 monoclonal (Stratagene), anti-PP2A C-subunit 1F6, and
anti-PP2A A-subunit (C-20, Santa Cruz Biotechnology) anti-
bodies and developed as described above.
GST fusion proteins were resolved by SDS-PAGE and trans-
ferred to a nitrocellulose membrane. The membrane was
washed three times in IMK buffer (10 m