1. Volume 4, Issue 4, Pages 748-758 (July 2011)
The Cyclophilin AtCYP71 Interacts with CAF-1 and LHP1 and Functions in Multiple Chromatin Remodeling Processes 
Li Hong , Luan Sheng  
Molecular Plant 
Volume 4, Issue 4, Pages (July 2011)
DOI: /mp/ssr036
Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

2. Figure 1 Physical Interaction of Arabidopsis AtCYP71 with LHP1.
(A) Scheme of the LHP1 and AtCYP71 protein domains used for interaction mapping. WD40, PPIase, chromo, and chromo shadow domains are used as representatives. Names of each construct are listed on the left. Numbers above the domains indicate amino acid positions in the full-length protein.
(B) Yeast two-hybrid analysis of AtCYP71 and LHP1. A dilution (1:10) series of yeast cells expressing the different combination of proteins were plated on the medium lacking leucine and tryptophan (−LT) or medium lacking leucine, tryptophan, and histidine (-LTH) and supplemented with 1 mM 3-AT. Growth of yeast cells on −LTH plates indicates positive protein–protein interaction.
(C, D) In vitro pull-down assays. LHP1–His-tagged proteins were immobilized on Ni-NTA agarose beads and incubated with GST fusion proteins. GST was used as a negative control. Western blots were performed using GST antibody. Names of GST fusion proteins are indicated at the bottom of each picture.
(E–L) Bimolecular fluorescence complementation (BiFC) analysis. (E–H), BiFC analysis of NE–AtCYP71 and LHP1–CE; (I, J), BiFC analysis of NE–AtCYP71 and CE (E, I), brightfield; (F, J), DIC; (G, K), GFP fluorescence images; (H, L), DAPI staining.
Molecular Plant 2011 4, DOI: ( /mp/ssr036)
Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

3. Figure 2
In Vitro Pull-Down Assays of Interaction between AtCYP71 and FAS1.
FAS1–His-tagged proteins were immobilized on Ni-NTA agarose beads and incubated with GST-tagged AtCYP71 or GST. Western blots were performed using GST antibody. Names of GST fusion proteins are indicated at the bottom of each picture.
Molecular Plant 2011 4, DOI: ( /mp/ssr036)
Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

4. Figure 3
Phenotypic Analysis of Double Mutant tfl2 cyp71 at Different Developmental Stages.
(A) Phenotype of an 18-day-old Col wild-type plant.
(B) Phenotype of an 18-day-old cyp71 plant.
(C) Phenotype of an 18-day-old tfl2 plant.
(D) Phenotype of a 36-day-old Col plant.
(E) Phenotype of a 36-day-old cyp71 plant.
(F) Phenotype of an 18-day-old tfl2 cyp71 double mutant (seedling lethal).
(G) Phenotype of a 36-day-old tfl2 plant.
(H) Phenotype of a 36-day-old tfl2 cyp71 double mutant.
(I) Phenotype of siliques from different plants (from left to right are Col, tfl2 cyp71, and tfl2 cyp71). Bars = 0.5 cm in (A–C) and (E–G). Bars = 0.2 cm in (D, H).
Molecular Plant 2011 4, DOI: ( /mp/ssr036)
Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

5. Figure 4
Genotyping and Histological Analysis of Double Mutant tfl2 cyp71.
(A) Scheme showing allele-specific RFLP to identify tfl2 cyp71 double mutant.
(B) DNA agarose gel showing AflII digestion pattern of DNA sample from plants of different genetic background.
(C–G) Cross-sections through an ovary of 45-day-old wild-type and mutants. (C), wild-type; (D), cyp71 mutant; (E), tfl2 mutant; (F, G), tfl2 cyp71 double mutants.
Bars = 0.3 mm in (C–G). Arrowheads indicate ovules.
Molecular Plant 2011 4, DOI: ( /mp/ssr036)
Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

6. Figure 5
ChIP Analysis of H3K27me3 Enrichment and LHP1 Association with Chromatin in cyp71 Mutant versus the Wild-Type.
(A) Western blots showing expression of LHP1–HA or LHP1–His in plants.
(B) Real-time PCR showing relative enrichment of LHP1–HA at the target genes in cyp71 compared with wild-type. ChIP results are expressed as fold change normalized to that of ACTIN and then to the value of the wild-type plants. The value of wild-type was arbitrarily set as 1 for normalization. Bars show standard error.
(C) ChIP assay of H3K27me3 enrichment at some LHP1 associated loci in mutant versus wild-type.
The abbreviations for these genes used in the figure are: AG, AGAMOUS; FT, FLOWERING LOCUS T; GY, GLYCOSYLASE; TB30, tb30g11; TAO1, ta01f06; TB36, tb36e06; 780, At4g19780; 790, At4g19790.
Molecular Plant 2011 4, DOI: ( /mp/ssr036)
Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

7. Figure 6
Gene Expression Analysis in tfl2 and cyp71 Single and Double Mutants.
(A) RT–PCR analysis of mRNA levels of representative genes associated with LHP1. Fourteen-day-old leaves from tfl2, cyp71, tfl2 cyp71, and wild-type plants were used to perform RT–PCR. The experiment was repeated three times using independent biological samples; one typical gel is presented.
(B) Real-time PCR analysis of relative mRNA levels in the mutants and the wild-type plants.
Molecular Plant 2011 4, DOI: ( /mp/ssr036)
Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

8. Figure 7
Class I KNOX Gene Expression Analysis in fas1 Mutant and Wild-Type.
(A) RT–PCR analysis of mRNA levels of class I KNOX genes. Twenty-five-day-old leaves from fas1, cyp71, and wild-type plants were used to perform RT–PCR. The experiment was repeated three times using independent biological samples. One typical gel is presented.
(B) RT–PCR analysis of mRNA levels of KNAT1 gene expression in fas1 and wild-type plant. Eigth-day-old leaves and 14-day-old leaves from fas1, cyp71, and wild-type plants were used to perform RT–PCR, respectively.
(C) Real-time PCR showing relative enrichment of FAS1–3FLAG at the KNAT1 gene in cyp71 compared with wild-type. ChIP results are expressed as fold change normalized to that of ACTIN and then to the value of the wild-type plants. The value of wild-type was arbitrarily set as 1 for normalization. Bars show standard error.
Molecular Plant 2011 4, DOI: ( /mp/ssr036)
Copyright © 2011 The Authors. All rights reserved. Terms and Conditions