H3 | Histone H3 (rabbit antibody) (nuclear marker)

Analysis of protein fractions from different steps of nuclei isolation. The protein fractions obtained following the main steps of the procedure used for isolation of nuclei from leaves of apple are shown. Equal amounts of proteins (4 ?g) from each fraction were loaded on the gel. A) Lumenal-binding protein 2 (BiP2), B) histone H3 and C) plastocyanin (PC) were detected with specific antibodies by western blot analysis. D) Coomassie blue staining of the fractionated proteins separated by polyacrylamide gel electrophoresis. Lane 1: crude extract of proteins from homogenized leaf tissue; lane 2: resuspended pellet of the whole cell lysate obtained following treatment with Triton X-100 and centrifugation; lane 3: nuclei collected from the 60% Percoll layer (see nuclear fraction in Figure 1B); lane 4: the interface fraction of 60% Percoll and 2.5 M sucrose layers in the density gradient containing chloroplasts and unbroken cells (see 60% P/2.5 M S interface in Figure 1B); lane 5: sucrose layer of the density gradient (see 2.5 M sucrose layer in Figure 1B); lane 6: nuclei purified by centrifugation on a 35% Percoll cushion.

Auxin stimulates PTRE1 accumulation at the plasma membrane.(a) Western blot analysis showed that auxin treatment results in relatively more PTRE1 at the plasma membrane, but decreased levels in nucleus and cytoplasm. Arabidopsis seedlings expressing PTRE1-GFP were treated with NAA (1??M, 60 or 120?min) and then surface-exposed membrane proteins were analysed using PTRE1 antibody or plasma membrane marker H+-ATPase. Nucleus and cytoplasm fractions were prepared from Col seedlings and analysed by western blot using antibody against PTRE1, nuclear marker H3 or cytoplasm marker UGPase. The relative quantities of the proteins were calculated by using image pro plus and indicated. (b) A proposed model for how PTRE1 and TIR1 coordinate auxin responses regulating proteasome activity and Aux/IAA protein degradation. Under normal condition (with basal auxin levels), 26S proteasome activity is maintained by appropriate distribution of PTRE1 at the plasma membrane and in intracellular compartments. In response to auxin, auxin rapidly stimulates the association of TIR1 and Aux/IAA proteins resulting in degradation of Aux/IAAs (1). Later, auxin suppresses PTRE1 to inhibit proteasome activity (possibly through stimulating the accumulation of PTRE1 at plasma membrane, resulting in decreased intracellular and nuclear localization) and hence suppresses Aux/IAA protein degradation (2) to coordinate regulation of auxin responses, reflecting a mechanism for fine control of Aux/IAA homeostasis and auxin signalling.

ths1 and vip3-2 mutants have altered global and genic histone H3 methylation patterns and fail to induce TCH3 and TCH4 in response to touch. (A) Chromatin extracts from cauline leaves of the indicated genotypes were separated by SDS-PAGE and detected with antibodies specific to H3K36me3 or H3K27me3. (B) Relative enrichment for H3K36me3 at the TCH3 and TCH4 loci in cauline leaf chromatin. Chromatin was isolated as in (A), then immunoprecipitated with the H3K36me3 antibody or without antibody and precipitated DNA amplified by qRT-PCR. Data presented is a percentage of the input, normalized to the S-ADENOSYLMETHIONINE SYNTHASE (SAM) gene and represented as a fold difference with the wild type non-treated sample. Error bars indicate the SEM for three biological replicates with two technical replicates each. (C) Quantitative reverse-transcriptase PCR analysis of TCH3 and TCH4 expression in response to touch in wild type and mutant seedlings. Seedlings were brushed for 2 minutes with a paintbrush according to the scheme shown in the top panel. RNA was prepared from the aerial tissues of treated and untreated plants and cDNA amplified with gene specific primers. The data was normalized to TUB4 (upper panels) or TCTP (lower panels) as reference genes and is presented as fold change compared to wild type non-treated samples. The error bars indicate SEM from three replicates. Asterisks mark significant difference from the untouched control P<0 05.

MYB3R3 accumulation in the root tip. a, c Confocal microscopy images of root tips. Five-day-old myb3r3 seedlings harbouring ProMYB3R3::MYB3R3-GFP were treated with 2??M zeocin for the indicated times a, or with 2??M zeocin, 50??M MG132 or 25??M roscovitine for 24?h c. Scale bars, 100??m. b, d Protein level of MYB3R3-GFP in roots. Ten-day-old myb3r3 seedlings harbouring ProMYB3R3::MYB3R3-GFP were treated with 5 or 10??M zeocin for 24 or 48?h b, or with 2??M zeocin, 50??M MG132 or 25??M roscovitine for 24?h d. Forty (b) or 20?µg (d) of total protein extracted from roots were subjected to immunoblotting using antibodies against GFP, ?-tubulin or histone H3. Protein extract from the myb3r3 mutant was used as a control (d). Relative levels of MYB3R3-GFP are expressed as the fold change, normalized with respect to the band of ?-tubulin or histone H3

Phosphorylation state of MYB3R3 is associated with protein accumulation and zeocin response. a Confocal microscopy images of myb3r3 root tips harbouring ProMYB3R3::MYB3R3AAA-GFP or ProMYB3R3::MYB3R3DDD-GFP. Five-day-old seedlings were treated with or without 2??M zeocin, or with 50??M MG132, for 24?h. Scale bars, 100??m. b Protein level of MYB3R3-GFP in roots. Ten-day-old myb3r3 seedlings harbouring ProMYB3R3::MYB3R3-GFP (WT), ProMYB3R3::MYB3R3AAA-GFP (AAA) or ProMYB3R3::MYB3R3DDD-GFP (DDD) were treated with or without 2??M zeocin for 24?h, and 40?µg of total protein extracted from roots was immunoblotted with anti-GFP or anti-histone H3 antibodies. Protein extract from myb3r3 was used as a control. Relative levels of MYB3R3-GFP are expressed as the fold change, normalized with respect to the band of histone H3. c Eighteen-day-old seedlings of WT, myb3r3 and myb3r3 carrying ProMYB3R3::MYB3R3AAA-GFP or ProMYB3R3::MYB3R3DDD-GFP. Scale bars, 2?cm. d, e Root growth of WT, myb3r3 and myb3r3 carrying ProMYB3R3::MYB3R3-GFP, ProMYB3R3::MYB3R3AAA-GFP or ProMYB3R3::MYB3R3DDD-GFP. Five-day-old seedlings were transferred to medium with or without 2?µM zeocin, and root length was measured for 6 days d. Root length after 6 days of zeocin treatment is shown in e. Data are presented as mean?±?SD (n?>?30). Significant differences from WT were determined by Student’s t-test: *P?

Reactant: Arabidopsis thaliana (Thale cress)

Application: Western Blotting

Pudmed ID: 29180698

Journal: Nat Commun

Figure Number: 4B

Published Date: 2017-11-27

First Author: Huot, B., Castroverde, C. D. M., et al.

Impact Factor: 13.783

Open Publication

Induction of NPR1 nuclear localization is retained at 30?°C. a Representative confocal microscopy images of NPR1-YFP plants (n?=?4) 24?h after spraying with mock or BTH. Images are of YFP (yellow) alone or YFP overlaid on Brightfield (BF, grey-scale). Scale bar length represents 10?µm. b Western blots of whole-cell lysate (W), non-nuclear (C, cytosolic) and nuclear (N) enriched fractions isolated from NPR1-Y1 transgenic plants treated with mock (?) or BTH (+) solutions at 23?°C or 30?°C. Equal volumes (10?µl) of each protein sample were loaded and run in two separate 4–12 % gradient SDS-PAGE gels. Following transfer to PVDF membranes, one blot was probed with ?-GFP primary antibody to detect the NPR1-YFP protein while the other blot was cut in two and the upper portion probed with ?-UGPase (a cytosolic protein control) and the lower portion probed with ?-H3 (a nuclear protein control). Whole-cell lysate extracted from npr1 plants treated with BTH at 23?°C was used as the negative control for the NPR1-YFP band. All data are representative of three independent experiments

Reactant: Botrytis cinerea

Application: Western Blotting

Pudmed ID: 32098836

Journal: mSystems

Figure Number: 1E

Published Date: 2020-02-25

First Author: Li, H., Zhang, Z., et al.

Impact Factor: None

Open Publication

Generation of ?bcactA mutants. (A) Replacement strategy for deletion of the bcactA gene. The 5? flank and 3? flank of bcactA were amplified and ligated into pLOB7 to flank the hygromycin resistance cassette to obtain the replacement vectors. Deletion mutants were generated by replacing the bcactA gene with the hygromycin resistance cassette through transformation of the protoplasts of the wild-type (WT) strain. (B) PCR diagnosis of ?bcactA mutants. PCR was performed using the primer pair bcactA-homo-up/HPH-det, to ensure that homologous recombination occurred at the target site. (C) Diagnosis of the homozygotes of ?bcactA mutants. PCR was performed with the primer pair bcactA-homo-up/bcactA-R-down, to ensure that the mutants were homozygous with no wild-type bands. (D) Southern blot analysis of WT and ?bcactA strains. The genomic DNA was digested with SacI and BamHI, separated in an agarose gel, and hybridized with a probe (a fragment on the hygromycin resistance cassette labeled with digoxigenin). Numbers represent different strains. (E) Immunoblotting analysis of BcactA protein in WT and ?bcactA strains. Total cytoplasmic proteins were extracted from 2-day-old mycelia cultured in PDB and immunoblotted with commercially available antibody to actin (Abmart; M20011). Histone 3 (H3) was used as a loading control.

Reactant: Arabidopsis thaliana (Thale cress)

Application: Western Blotting

Pudmed ID: 32210987

Journal: Front Plant Sci

Figure Number: 3A

Published Date: 2020-03-27

First Author: Fu, C., Liu, X., et al.

Impact Factor: 5.435

Open Publication

Western blot results exhibiting the expression of BRF2 protein in 10-day-old seedlings of WT and fes1a mutants.

Reactant: Arabidopsis thaliana (Thale cress)

Application: Western Blotting

Pudmed ID: 34194459

Journal: Front Plant Sci

Figure Number: 5A

Published Date: 2021-07-02

First Author: Noh, S. W., Seo, R. R., et al.

Impact Factor: 5.435

Open Publication

Monomethylation of histone H3K4 is associated with the transcription of genes encoding transcription factors in Arabidopsis. (A) Abundance of H3K4me1, H3K4me2, and H3K4me3 marked proteins in WT and ldl1 ldl2 plants. The right panel shows modified histone H3 protein levels in Arabidopsis (average ± SD, *p < 0.05, Student’s t-test, n = 3). The results of all independent experiments are shown in Supplementary Figure 6. (B) Genomic DNA regions of gene-specific primers used in panels (C-E). (C-E) ChIP-qPCR to examine the enrichment of modified histone H3 proteins in the identified LDL1-target genes (C), transcription factors overrepresented in the ldl1 ldl2 mutants (D), and PR1 and FRK1 genes (E). Enrichment of Lys 4- mono-, Lys 4- di-, and Lys 4-trimethylated histone H3 protein at the chromatin regions of genes was measured in wild type (white bars) and ldl1 ldl2 (gray bars). The amount of DNA after ChIP was quantified by qPCR, and the means represent the average immunoprecipitation efficiencies (%) against total input DNA used. Each plot shows a representative of two independent biological replications (with similar results), and the data points are the average values of technical triplicates (average ± SEM, *p < 0.05, Student’s t-test, n = 3). The blue bars are the amplicon regions used for ChiP-qPCR, and the primer sequences used in this study are shown in Supplementary Table 2.