Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2020 Sep 25;11:4859. doi: 10.1038/s41467-020-18600-8

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

PMC Copyright notice

Fig. 4 — a LET2/MDS1 associates with MEKK2 and SUMM2, but not MPK4. LET2-HA was co-expressed with Ctrl, MEKK2-FLAG, SUMM2-FLAG, or MPK4-FLAG in protoplasts for 12 h. The FLAG-tagged proteins were immunoprecipitated by α-FLAG affinity beads, and then immunoblotted by an α-HA or α-FLAG antibody (top two panels). The proteins before immunoprecipitation were immunoblotted by an α-HA or α-FLAG antibody as inputs (bottom two panels). b MEKK2 stabilizes LET2/MDS1 protein accumulation in N. benthamiana. LET2-HA or GFP was co-expressed with MEKK2-GFP or GFP in N. benthamiana for 3 days. The proteins were immunoblotted by an α-HA or α-GFP antibody. CBB staining of RBC was used as a loading control. c MG132 treatment increases LET2/MDS1 protein accumulation in transgenic plants. The 10-day-old seedings of 35S::LET2-HA/let2-1 (Line #2 and #3) transgenic plants were treated with DMSO (Ctrl) or 5 μM MG132 for 6 h. Proteins were immunoblotted using an α-HA antibody, and CBB was used as a loading control. d MG132 treatment increases LET2/MDS1 protein accumulation in N. benthamiana. The leaves of N. benthamiana were inoculated with Agrobacterium carrying LET1-HA and GFP, or LET1-HA and MEKK2-GFP for 12 h, and then treated with DMSO (Ctrl) or 5 μM MG132 for anthor 36 h. Proteins were immunoblotted by an α-HA or α-GFP antibody. CBB was used as a loading control. e Silencing MEKK1 increases LET2/MDS1 protein accumulation. MEKK1 was silenced in the 35S::LET2-HA/let2-1 transgenic plants (Line #1, #2 and #3) by VIGS. Total proteins were extracted 2 weeks after VIGS, and immunoblotted using an α-HA antibody. CBB was used as a loading control. f LET2/MDS1, but not its kinase-inactive mutant LET2^KM, induces LET1 mobility shift. LET2-HA or LET2^KM-HA was co-expressed with LET1-FLAG in protoplasts for 12 h. LET1-FLAG was separated by 7.5% SDS-PAGE. CBB staining of RBC was used as a loading control. g LET2/MDS1 induces LET1 phosphorylation. LET1-HA was co-expressed with Ctrl or LET2-FLAG in protoplasts for 12 h. LET1-HA was immunoprecipitated by α-HA affinity beads. The immunoprecipitated LET1-HA protein was incubated without or with 0.5 μL (200 U) λ-phosphatase (Sigma) for 1 h at 30 °C. LET1-HA was separated by 10% SDS-PAGE and detected by an α-HA antibody (top panel). LET1-HA and LET2-FLAG before immunoprecipitation were detected by the corresponding antibody (middle two panels). CBB staining of RBC was used as a loading control (bottom panel). h LET2/MDS1 increases LET1 kinase activity. LET1-FLAG or LET1^KM-FLAG was co-expressed with the vector control, LET2-HA or LET2^KM-HA, in protoplasts. The FLAG-tagged proteins were immunoprecipitated from the cell lysates with α-FLAG affinity beads and used in a kinase assay with [γ-³²P] ATP. The GFP-FLAG was used as a negative control. The proteins were immunoblotted by an α-FLAG or α-HA antibody for input controls. i LET1 associates with LET2/MDS1. LET1-HA was co-expressed with Ctrl or LET2-FLAG in protoplasts for 12 h. The LET2-FLAG proteins were immunoprecipitated by α-FLAG affinity beads, and then immunoblotted by an α-HA or α-FLAG antibody (top two panels). The proteins before immunoprecipitation were immunoblotted by an α-HA or α-FLAG antibody as inputs (bottom two panels). j, k FRET-FLIM analysis of LET1 and LET2/MDS1 interaction in Arabidopsis protoplasts. The indicated proteins were transiently expressed in protoplasts for 16 h, and FRET-FLIM was visualized using a confocal laser scanning microscopy (j). Localization of the LET1-GFP and LET2-mCherry/BIR2-mCherry is shown with the first (Green) and second column (Red), respectively. The lifetime (τ) distribution (third column), and apparent FRET efficiency (fourth column) are presented as pseudo-color images according to the scale. The GFP mean fluorescence lifetime (τ) values, ranging from 2.2 to 2.7 nanoseconds (ns), were statistically analyzed and are shown as mean ± SD (n = 15) (k). P = 1.07 × 10⁻¹² (column 1 and 2), P = 1.08 × 10⁻¹² (column 2 and 3). The different letters indicate the significant difference determined by one-way ANOVA followed by the Tukey test (P < 0.05). Scale bar, 10 µm. l LET2^ex associates with LET1 in a pull-down assay. Arabidopsis protoplasts expressing LET1-FLAG were incubated with purified HIS-SUMO-LET2^ex proteins. The interaction between LET1 and LET2^ex was detected by an α-FLAG immunoblot after pull-down with Ni-NTA agarose. HIS-SUMO-LET2^ex proteins were stained by CBB. The above experiments were repeated three times with similar results.