Supporting Information
Cytokinin signaling stabilizes the response activator ARR1
Jasmina Kurepa, Yan Li and Jan A. Smalle*
Figure S1. Specificity of the anti-ARR1antibodies.
Figure
S2.
Analyses
of
the
relationship
between
protein
abundance
and
chemiluminescence signal intensity on immunoblots probed with anti-ARR1 sera.
Figure S3. Effects of MG132 and translation inhibitor treatments on total proteins and on
ARR1.
Figure S4. Effects of four-hour-long CHX and MG132 treatments on the ARR1 transcript
level.
Table S1: Primers used in this study
1
Figure S1. Specificity of the anti-ARR1antibodies.
(a)
Alignment of the ARR1 peptide antigen sequence with the corresponding region
of the ARR2 protein. Sequence alignment was done using PRALINE (Simossis and
Heringa 2005). Phylogenetic analyses of the RRB genes have shown that ARR1 and
ARR2 belong to same subfamily (Mason et al. 2004). The ARR1 peptide antigen was
chosen from the region that has minimal homology with ARR2.
(b)
Analysis of the ARR1 level in the wild type Col-0 and in the arr1-1 mutant (Sakai
et al. 2001). The molecular mass of the protein standards (M; Precision Plus Protein™
Prestained Standards, http://www.bio-rad.com) is noted on the left-hand side.
Immunoblot (IB) with anti-ARR1 antibodies is presented using Multi-Channel Viewer
(Quantity One software, Bio-Rad). The region of the Ponceau S stained membrane with
the large subunit of RuBisCO is shown to illustrate loading.
(c)
The anti-ARR1 antibody does not recognize the ARR2 protein. Six-day-old wild-
type (Col-0), arr1-1, arr2-3 [(Mason et al. 2005); ABRC stock CS6974] and arr2-4
[(Mason et al. 2005); ABRC stock CS6975] seedlings were used to prepare total protein
extracts. Immunoblot with anti-tubulin (TUA) antibodies is shown as a loading control.
(d)
In six-day-old seedlings, the alternatively spliced form At3g16857.1, which
encodes a 669 amino acid-long protein, is more abundant than At3g16857.2 that
encodes a 690 amino acid-long protein. Relative transcript levels were determined using
qPCR analyses with GADPH as a reference gene essentially as described (Li et al.
2013). The ARR1-specific primer sequences are presented in the Table S1. The
experiment was done using two biological replicates with three technical replicates each.
The transcript level of ARR1.1 was assigned the value of 1. These data suggest that the
higher molecular weight ARR1 isoform may represent the protein encoded by
At3g16857.2.
Figure
S2.
Analyses
of
the
relationship
between
protein
abundance
and
chemiluminescence signal intensity on immunoblots probed with anti-ARR1 sera.
(a)
ARR1 immunoblotting analysis of a serial dilution of total protein extract from
six-day-old Col-0 seedlings. Anti-RRA1 antibody was used at a dilution of 1:10,000 and
2
the secondary antibody (goat anti-rabbit IgG-HRP; sc-2030, Santa Cruz Biotech) was
used at 1:2500. Chemiluminescent signals were detected using SuperSignal West
Femto Chemiluminescent Substrate (Thermo) and a CCD camera (ChemiDoc, Bio-Rad).
(b)
Signal intensity vs. protein amount graph showing the best-fit linear regression.
Signal strengths were quantified using QuantityOne software (Bio-Rad). The mean of
relative intensity and the standard error of mean (n=2 blots with two technical repetitions
per blot) are shown.
Conclusion: The relationship between protein abundance and chemiluminescence signal
intensity was linear when 5 - 20 µl of the protein extract (obtained as presented in the
Experimental methods section) was loaded per lane. In experiments described in this
study, 15 µl of total extract was loaded per lane.
Figure S3. Effects of MG132 and translation inhibitor treatments on total proteins and on
ARR1.
(a)
Treatment control for Figure 2a. Six-day-old seedlings grown on MS/2 media
were treated for 4 hours DMSO (solvent control) or 100 µM MG132. Total protein
extracts were separated on 10% gels, blotted and the immunoblot (IB) was probed with
1:1000 diluted antibodies against polyubiquitinated (polyUb) species obtained from Enzo
Life Sciences (http://www.enzolifesciences.com/).
(b)
ARR1 stability after puromycin treatments. Puromycine (PUR) covalently
attaches to the C-terminus of the nascent polypeptide chains causing their premature
termination. Six-day-old seedlings were treated for the denoted time with 100 µM PUR
and used for the extraction of total proteins. Proteins were separated on 7.5% gels.
Membranes were probed with either anti-ARR1 or monoclonal anti-PUR antisera
(working dilution 1:10,000; Millipore, http://www.millipore.com/).
(c)
Long-term CHX treatments. Six-day-old seedlings were treated with CHX for the
denoted time. Total protein extracts were separated on 7.5% gels, blotted and probed
with anti-ARR1 antibodies. The same membrane was re-probed with anti-TUA
antibodies.
3
(d)
CHX dose-response. Six-day-old seedlings were treated with high CHX doses for
2 hours. The IB was done as in (c).
Figure S4. Effects of four-hour-long CHX and MG132 treatments on the ARR1 transcript
levels.
Plants grown for five days on MS/2 media were treated either with DMSO (solvent
control), 200 µM CHX or 100 µM MG132 for 4 hours. RNA isolation, cDNA synthesis and
qPCR were done as previously described (Li et al. 2013). The sequence of the ARR1specific primers designed to amplify both alternatively spliced transcripts is listed in
Table S1. The average relative ARR1 levels of two biological replicates (with three
technical replicates each) are shown. Error bars are SD.
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