1. Allele-Specific Suppression of a Defective Brassinosteroid Receptor Reveals a Physiological Role of UGGT in ER Quality Control 
Hua Jin, Zhenyan Yan, Kyoung Hee Nam, Jianming Li 
Molecular Cell 
Volume 26, Issue 6, Pages (June 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1 ebs1 Suppresses bri1-9 Phenotype by Affecting BR Perception
(A) Schematic presentation of the genetic screen for ebs mutants in the bri1-9 background (see the Experimental Procedures for details).
(B) Four-week-old soil-grown plants of bri1-9, wild-type, and ebs1-1 bri1-9.
(C) Seven-week-old mature plants of bri1-9, wild-type, and ebs1-1 bri1-9 grown in soil.
(D) The hypocotyl length of 4-day-old dark-grown seedlings of bri1-9, wild-type, and ebs1-1 bri1-9. Each bar represents the average measurement of ∼40 seedlings of two duplicate experiments. Error bar denotes standard error of the mean.
(E) bin2-1/+, bin2-1, and their corresponding double mutants with ebs1-1 grown in soil for 6 weeks.
(F) det2 and ebs1-1 det2 mutants grown in soil for 6 weeks.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 ebs1 Restores BR Sensitivity of bri1-9
(A) Quantitative analysis of BR sensitivity of bri1-9, wild-type, and ebs1-1 bri1-9 seedlings by the root inhibition assay as described previously (Li et al., 2001). Each data point represents the average root elongation of ∼40 seedlings of two duplicate experiments. Error bar denotes standard error of the mean.
(B) BR-induced changes in the phosphorylation status of BES1. Three-week-old seedlings of bri1-9, wild-type, and ebs1-1 bri1-9 were treated with 1 μM BL for 1 hr in liquid half MS medium. Total proteins were extracted with 2× SDS buffer and separated by a 10% SDS-PAGE. BES1 was detected by anti-BES1 antibody (Mora-Garcia et al., 2004). Nonspecific bands (∗) were used as a loading control in the lower panel.
(C) Feedback inhibition of CPD expression by BL. Total mRNAs were extracted from BL-treated seedlings and probed for CPD as described previously (Li et al., 2001). The filter was stripped and reprobed with the 18S rDNA for loading control.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 ebs1 Is an Allele-Specific Suppressor of the bri1-9 Mutation
(A) ebs1 cannot suppress bri1-301 and bri1-101 mutants containing a kinase domain mutation. From left to right: 6-week-old soil-grown plants of bri1-301, ebs1-1 bri1-301, bri1-101, and ebs1-3 bri1-101.
(B) ebs1 fails to suppress bri1-6 and bri1-113 mutants harboring mutations in the BR binding domain. From left to right: 5-week-old soil-grown plants of bri1-6, ebs1-1 bri1-6, bri1-113, and ebs1-1 bri1-113.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

5. Figure 4 Molecular Characterization of the EBS1 Gene
(A) Genetic mapping of EBS1. EBS1 was mapped to a 63 kb region at the bottom of chromosome I between markers CER and F3I17_2, which is covered by two overlapping BAC clones: F23N20 and F3I17. Molecular makers and numbers of recombination for each marker are shown above and below the line, respectively. Sequence analysis of the entire 63 kb region of ebs1-1 identified a single-nucleotide mutation in At1g71220 composed of 37 exons (bar) and 36 introns (line). Arrows indicate the positions of five ebs1 mutations.
(B) Summary of five ebs1 alleles, their predicted molecular defects, and the corresponding positions in the human UGGT (accession number Q9NYU2).
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
bri1-9 Is Retained in the ER but Moves to the PM in ebs1 Mutants
(A) Endo H sensitivity of BRI1 and bri1-9. Total proteins from bri1-9, wild-type, and ebs1-1 bri1-9 to ebs1-5 bri1-9 were subjected to Endo H treatment followed by immunoblotting with anti-BRI1 antiserum.
(B–D) Western blotting analysis of membrane fractions obtained through aqueous two-phase partitioning of total microsomal proteins isolated from bri1-9 (B), wild-type (C), and ebs1-3 bri1-9 (D) plants using antibodies against BRI1, BiP, PDI, PMA2, and PIP2. M, U, and L indicate total microsomal fraction, the upper phase that enriches PM proteins, and the lower phase that was depleted of PM, respectively.
(E–M) Confocal analysis of subcellular localization of BRI1:GFP and bri1-9:GFP in root tips of 6-day-old light-grown seedlings coexpressing DsRed:HDEL and BRI1:GFP (E–G) or bri1-9:GFP in either EBS1+ (H–J) or ebs1 (K–M) background.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 The ebs1 Mutation Inhibits Monoglucosylation of bri1-9
(A) Schematic illustration of the JBαM action. Hexagon, circle, and diamond represent glucose, mannose, and N-acetylglucosamine residues, respectively.
(B) Sensitivity of BRI1:GFP and bri1-9:GFP to JBαM treatment. Anti-GFP immunoprecipitates of BRI1:GFP, bri1-9:GFP, and bri1-9:GFP ebs1-3 transgenic plants were incubated with or without JBαM followed by western blotting analysis with an anti-GFP antibody.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7 bri1-9 Interacts with CNXs and BiPs
Coimmunoprecipitation of bri1-9:GFP with BiPs and CNXs. Total protein crude extracts from wild-type plants (lane 1) or transgenic plants of BRI1:GFP (lane 2), bri1-9:GFP (lane 3), or bri1-9:GFP ebs1-3 (lane 4) were immunoprecipitated with anti-GFP antibody and analyzed by western blotting with antibodies against GFP, a maize CRT, or BiP. The left and right four lanes show the presence of BRI1:GFP/bri1-9:GFP, CNXs, CRTs, and BiPs in total protein extracts and anti-GFP immunoprecipitates, respectively. The numbers represent the relative signal intensity of coimmunoprecipitated CNXs or BiPs after normalization against the anti-GFP signal.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions