SUPPLEMENTAL FIGURE LEGENDS
Figure S1. Characterization of p27Kip1 pattern by 2D/WB
Panel A: 200 μg protein of a nuclear extract of Lan-5 cells were
immunoprecipitated
with
polyclonal
antibodies
against
pS10-p27Kip1
(pS10p27). Half of pS10-p27Kip1 IP was incubated with a protein phosphatase
(λ PPase) (bottom). The other half was treated identically but without the
enzyme (top). The samples were analyzed by 2D/WB and the immunoblotting
were performed employing mAbs against p27Kip1.
Panel B: 200 μg protein of a nuclear extract of Lan-5 cells were
immunoprecipitated
with
polyclonal
antibodies
against
pS10-p27Kip1
(pS10p27). From the top to the bottom, Input (INPUT); supernatant after
immunoprecipitation (SN IP pS10p27), and immunoprecipitated materials (IP
pS10p27). The immunoblotting was performed employing mAbs against
p27Kip1.
Panel C: Lan-5 cells were transfected with identical amount of wild-type
pcDNA3-p27Kip1 and pcDNA3-p27Kip1-S10A. After 24 hours, cells were
collected and nuclear extracts prepared. 200 μg of proteins were analyzed by
2D/WB employing monoclonal antibodies against p27Kip1 (WB p27).
Panel D: Immunoblotting analysis of the two-dimensional gel electrophoresis of
HeLa (top) and human mesenchymal stem cells (hMSC) nuclear extracts (200 μg
protein each). The immunoblotting analyses were performed employing mAbs
against p27Kip1.
Panel E: K562 cells were cultured (or not) in the presence of imatinib 1 µM (IM)
for 24 hours. Then 500 µg extract from untreated cells (top) and imatinibtreated K562 cells were separated by 2D/WB and analyzed with mABs against p
p27Kip1 (images on the left). The same blots were analyzed with pABs against
pS10- p27Kip1 (images on the right).
Figure S2. Synchronization of Lan-5 cells and evaluation of efficiency of
anti pT187-p27Kip1 antiserum
Panel A. Lan-5 cells were synchronized as reported under Materials and Methods.
Then, cells were detached and the suspension analyzed by fluorescent activated cell
sorter for DNA content. The histogram reports the percentage of cells in G1, S and
G2/M phases.
Panel B. Lan-5 cells were synchronized as reported under Materials and Methods.
G0, G1, S and G2/M nuclear cell extracts were analyzed for HDAC1 (histone
deacetylase 1) and cyclin A content by immunoblotting
(top). HDAC1 was
employed for confirming equal loading. The same samples were analyzed for the
content in cyclin D1, E and B1 (bottom). The cyclins content reported here is an
example of materials employed in the study.
Panel C. To verify the efficiency of anti-pT187-p27Kip1 serum employed in IP
analysis, the following experiment was performed. Recombinant active
cyclinE/CDK2 was incubated with recombinant unmodified p27Kip1 or
recombinant p27Kip1-T187A. After 1 hour of incubation, the assay was stopped
by heating at 80°C for 5 min (p27Kip1 is an heat stable protein) and the antipT187-p27Kip1 serum was added. Then, we purified the immunocomplexes and
analyzed the immunoprecipitating material (IP) and the supernatants (S/N) for
p27Kip1 content.
Figure S3. p27Kip1 half-life characterization by 2D/WB pattern and p27Kip1
binding with CDK1
Panel A. Lan-5 cells were treated (or not) with 36 μM cycloheximide for 8 hours.
Then, cells were collected and nuclear extracts prepared. 200 μg proteins were
separated by 2D electrophoresis, blotted onto nitrocellulose and analyzed by
means of monoclonal antibodies against p27Kip1 (blots on the left) and
polyclonal antibodies against pS10-p27Kip1 (blots on the right).
Panel B. 2 mg of G2/M nuclear extracts were immunoprecipitated with
polyclonal antiserum against CDK1. Left: 500 μg of input extract were separated
by 2D/WB and probed with mAbs against p27Kip1. Right: The IP materials was
then analyzed by 2D/WB and probed with mAbs against p27Kip1.
Panel C. Equal amounts (1 mg) of asynchronous nuclear Lan-5 cell extract and
nuclear extract obtained from G1, S and G2/M phases were immunoprecipitated with
polyclonal antibodies against p27Kip1, pS10-p27Kip1 and pT187-p27Kip1. An
unrelated antiserum (NR) was employed as control. The filter was probed with mAbs
against p27Kip1 or CDK1. Note that the p27Kip1 blot was the same reported in Figure
5A.
Figure S4. Characterization of Skp2-bound p27Kip1 isoforms and
phosphotyrosine p27Kip1 isoforms
Panel A. 2 mg nuclear extract of Lan-5 cells were immunoprecipitated
employing antibodies against Skp2. Then, the IP materials (IP SKP2) and 200 μg
of nuclear extract (INPUT) were analyzed by 2D/WB using a monoclonal
antibodies against p27Kip1 (WB p27).
Panel B. Lan-5 cells were cultured for 8 hours in the presence of MG132 (1 μM)
to increase pT187-p27Kip1 and a nuclear extract was prepared. Two aliquots of
the extract (500 μg each) were immunoprecipitated: one with antibodies against
pS10-p27Kip1 and the other with an antibodies against pT187-p27Kip1. The two
supernatant were immunoprecipitated with antibodies against Skp2. Finally, the
two IPs (one shown as pS10-p27 DEPLETED MG132-TREATED EXTRACT and
the other as pT187-p27 DEPLETED MG132-TREATED EXTRACT) were analyzed
by 2D/WB using mAbs against p27Kip1 (WB p27).
Panel C. 2 mg nuclear extract of growing Lan-5 cells were immunoprecipitated
by antibodies against phosphotyrosine antibodies. Then, 2D/WB was performed
on the IP materials and the filter analyzed by monoclonal antibodies against
p27Kip1. From the top to the bottom: 100 μg input extract (INPUT); supernatant
of 100 μg protein of immunoprecipitated extract (100 μg SUPERNATANT IP PY),
IP from 2 mg nuclear extract (IP PY from 2 mg INPUT).
Panel D. 800 μg asynchronous nuclear Lan-5 nuclear extract were
immunoprecipitated using polyclonal antibodies against pS10-p27Kip1 and the
IP materials divided into two aliquots. One aliquot was incubated with a protein
tyrosine phosphatase (IP pS10p27 + LAR), the other aliquot was the control of
reaction (IP pS10p27 Cont). After one hour of incubation, the assay mixtures
were analyzed by 2D/WB using a monoclonal antibodies against p27Kip1. The
filter on the top is the untreated control IP (IP pS10p27 Cont) while the filter on
the bottom was the analysis of LAR-treated IP (IP pS10 + LAR).
Panel E. 1 mg aliquots of MG132-treated nuclear Lan-5 extract were
immunoprecipitated by: antibodies against pS10- p27Kip1 (IP pS10p27, first
lane); polyclonal antibodies against total p27Kip1 (IP p27, second lane); not
related polyclonal antibodies (IP NR, fourth lane). Moreover, the supernatant of
extract after IP with anti-pS10 p27Kip1 (reported as SN IP pS10p27) was
immunoprecipitated by polyclonal antibodies against total p27Kip1 (IP p27,
third lane). Each IP was analyzed by monodimensional immunoblotting
employing antibodies against p27Kip1 (WB p27) or phosphotyrosine protein
(WB pY). In the last case, the filter was exposed overnight.
Figure S5. Analysis of CDK associated to cyclinA and of cytosolic p27Kip1
isoforms. Characterization of p27Kip1 isoforms during the release from
ATRA-dependent cell cycle arrest
Panel A. Untreated nuclear extract and CDK2-depleted nuclear extract were
immunoprecipitated with anti-cyclin A antiserum and the content of p27Kip1 and
CDK1
was
estimated.
Additionally,
untreated
nuclear
extract
was
immunoprecipitated with anti-CDK2 antiserum and the content of p27Kip1 and
CDK1 was estimated.
Panel B: Lan-5 cells were synchronized as reported under Materials and Methods
and cytosolic extracts were prepared. Then, extracts of each phase (G0, G1, S and
G2/M) were analyzed by 2D/WB for p27Kip1 isoforms. Note that different amounts
of extract of each phase were employed in the experiment due to the difference of
p27Kip1 content. Particularly, 500 µg for the G0 and G1 phase analyses, 4 mg for S
phase analysis and 2 mg for G2 phase analysis.
Panel C: Lan-5 cells were treated for 48 hours with 5 μM retinoic acid (ATRA) to
allow the arrest of cells in G0/G1 (reference 46). Then, cells were released from
ATRA block and pelleted after 8 hours. An additional experiment was performed
by contemporaneously removing ATRA and adding MG132 (a proteasome
inhibitor) for 8 hours. Nuclear extracts were prepared: 1) before ATRA
treatment (control); 2)
after 48 hours ATRA treatment (+ ATRA 48 hours); 3) after 48 hours ATRA
treatment followed by 8 hours ATRA removal (- ATRA 8 hours), and 4) after 48
hours ATRA treatment followed by 8 hours ATRA removal (- ATRA 8 hours) and
MG132 addition. It is evident an increase of monophosphorylated p27Kip1 after
48 hours treatment and its decrease after 8 hours of ATRA removal. The specific
decrease is not modified by MG132, thus suggesting that it is not due to CDK2dependent p27Kip1 removal.
Figure S6. Characterization of 2D/WB p27Kip1 mutants isoforms.
Panel A. Lan-5 cells were transfected with 3 distinct expression vectors, namely
pcDNA3-p27Kip1-T187A, pcDNA3-p27Kip1-T187E, and pcDNA3-p27Kip1-S10E.
After 24 hours, cells were collected and the nuclear extracts were prepared. 200
μg nuclear extracts were then analyzed by 2D/WB employing antibodies against
p27Kip1. Note that the substitution of threonine 187 with a glutamic acid causes
a shift of the pattern towards a more acidic isoelectric point.
Panel B. 1 mg transfected nuclear extracts of Lan-5 (see panel A) were p27Kip1T187E transfected cells was immunoprecipitated with polyclonal antibodies
against pS10 p27Kip1 (IP pS10p27) while the extract from from pcDNA3p27Kip1-S10E-transfected cells employing the antibodies against pT187
p27Kip1 (IP pT187p27). The two IPs were then analyzed by 2D/WB employing
antibodies against p27Kip1.