Supporting Information Table S1 & Methods S1
Table S1 Pairs of primers used in RT-PCR or cloning
Gene
NtE2F
Name
Primer 3
Use
cloning
TSO2F
TSO2R
5’ 3’ sequence
GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGCAGC
AGCCACATCATCAACAGCGTGATGAGAGAGTGCATTTC
GATCCAA
GGGGACCACTTTGTACAAGAAAGCTGGGTTCAGCTCCC
AGTTGTATTTGCAGAAGGCAG
GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGCAGC
AGCCACATCATCAACAGCGTGATGAGAGAGTGCAT
GGGGACCACTTTGTACAAGAAAGCTGGGTGTTCAATTG
AA ACCTGATGTTGA
TCGCTTGTCTACTCTACACG
CCGCGTCGCAGACGATTGA
H2AxaF
H2AxaR
GATTTCTTCAAACTCTCATCAAATCG
GATTCGTTCCGAAATATCATATC
RT PCR
RT PCR
H2AxbF
H2AxbR
ACTCACAAAATCCTCAGCCATC
GAGGCTCATATCAAATGGGCTG
RT PCR
RT PCR
Primer 1
CTCGAGATGAGAGAGTCCCTTTGAATGGAGGAGTTTTG cloning
CCAAAGATCTTACCTGACCACACAG
ACTAGTAACGAGAGAGTACTGAGAATGGAGGAGTTTTG cloning
CCAAAGATAATCTAGAGAGAATAATG
Primer 4
Truncated Primer 5
NtE2F
Primer 6
TSO00000
02
(At3g2706
0)
H2AXa
(At1g0888
0)
H2AXb
(At1g5469
0)
miH2AX
Primer 2
cloning
cloning
cloning
RT PCR
RT PCR
Methods S1 Preparation of nuclear extracts and two-dimensional electrophoresis.
All steps were performed at 4°C. Mid-log phase BY-2 cells were harvested and washed three
times in PBS buffer (140 mM NaCl, 2.7 mM KCl, 1.5 mM K2H2PO4, 6.5 mM Na2HPO4 pH
7). Pelleted cells were ground in liquid nitrogen and thawed in 10 vol. of NGB buffer (1M
hexylene glycol, 10 mM PIPES/KOH pH 7, 10 mM MgCl2, 0.2% Triton x 100 , 5mM ßmercaptoethanol, 1mM PMSF). The suspension was filtered through a nylon membrane (50
µm mesh) and the filtrate was centrifuged at 2000g for 10 min. An aliquot of the supernatant
was centrifuged at 21000 g for clarification and kept as the cytosolic fraction. The initial
pellet was washed two times in 2 vol. of NGB2 buffer (NGB buffer with only 0.5 M hexylene
glycol) by centrifugation at 3000g for 30 min. The pellet was then resuspended in 0.5 vol. of
NLB buffer (100 mM KCl, 15 mM HEPES/KOH pH 7.5, 5 mM MgCl2, 1 mM DTT, 1 mM
PMSF) and 0.5 M (NH4)2Fe(SO4)2 , 6H2O for 30 min under shaking. Then after a
centrifugation of 5600 g the supernatant was dialyzed two times in HMK buffer (25 mM
HEPES/KOH pH 7.5, 50 mM KCl, 10% glycerol, 1 mM PMSF, 5mM MgCl 2, 0.25 µg/mL
pepstatine, 0.5 µg/mL Leupeptine).
Two-dimensional electrophoresis was essentially performed according to the manufacturer’s
instructions (Bio-Rad, Hercules, CA, USA). For the first dimension, ReadyStrip ™ IPG (BioRad) were actively rehydrated (8-10 h at 50 V) in 300 µL buffer (8 M urea, 2% CHAPS, 0.5%
ampholytes Bio-Lyte® [Bio-Rad] pH 3-10, 0.001% Bromophenol Blue) containing 1 to 2 mg
proteins. Then protein migration was performed as follows : 1 h at 250 V, then submitted to
increased voltage reaching a maximum of 10 000 V for 2–5 h and finally 1 h at maximum
voltage. During all the migration, the current limit was set at 50 µA per strip.
After focusing, the strips were balanced for15 min at 20°C in a reducing buffer (6 M urea, 2%
SDS, 0.375 M Tris-HCl pH 8.8, 20% glycerol, 2% DTT) and 15 min at 20°C in an alkylation
buffer (6 M urea, 2% SDS, 0.375 M Tris-HCL pH 8.8, 20% glycerol, 2.5% iodoacetamide,
0.001% Bromophenol Blue). The strips were then applied to 10% SDS-PAGE. The gels were
overlaid with 1% agarose solution to ensure good contact between the strip and the gel.