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. 2018 Nov 14;38(1):e99819. doi: 10.15252/embj.201899819

Figure 2. OST1 interacts with EGR2 and inhibits OST1 activity under cold stress.

Figure 2

  • A
    Interaction of OST1 and EGR2 in yeast. Yeast cells harboring different vector combinations were grown on SC/−Leu/−Trp medium for 3 days or SC/−Leu/−Trp/−His/−Ade medium for 6 days. OST1‐BD/AD and BD/EGR2‐AD were used as negative controls.
  • B
    OST1 interacts with EGR2 in vitro. Purified recombinant MBP‐His‐EGR2 or MBP‐His proteins from E. coli were immunoprecipitated with MBP beads and then incubated with purified recombinant GST‐OST1. Precipitated proteins were detected with anti‐His and anti‐GST antibodies.
  • C
    Co‐IP assay of OST1 with EGR2 in vivo. 35S:HF‐OST1/Super:EGR2‐Myc or 35S:HF‐OST1/Super:Myc was expressed in N. benthamiana leaves. Total proteins were immunoprecipitated with anti‐Myc agarose beads, and the co‐immunoprecipitation products were subjected to immunoblot analysis. EGR2‐Myc and HF‐OST1 were detected with anti‐Myc and anti‐HA antibodies, respectively.
  • D
    Bimolecular fluorescence complementation (BiFC) assay. Wild‐type Arabidopsis protoplasts transformed with OST1‐YFP C and EGR2‐YFP N were incubated for 18 h. The interaction signal was detected by confocal microscopy. The combinations of OST1‐YFPC/GUS‐YFPN and GUS‐YFPC/EGR2‐YFPN were used as negative controls. Scale bars: 100 μm.
  • E
    EGR2 inhibits OST1 kinase activity in vitro. Proteins were incubated with 1 μCi [γ‐32P]ATP in kinase reaction buffer for 30 min at 30°C and then separated by SDS–PAGE. Top, autoradiograph; bottom, CBB staining.
  • F, G
    In‐gel assay of OST1 activity in 10‐day‐old wild‐type, egr1 and egr2 single mutants, and the egr1 egr2 double mutant under cold stress.
  • H, I
    In‐gel assay of OST1 activity in 10‐day‐old wild‐type and EGR2‐overexpressing plants under cold stress.
Data information: In (F–H), the ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1.00. In (F and H), representative pictures are shown: Top, autoradiograph; bottom, CBB staining. In (G and I), relative kinase activity is shown. Each bar represents the mean ± SEM of three biological repeats. **P < 0.01 (two‐tailed t‐test).Source data are available online for this figure.