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

Figure 5. Cold stress disrupts the interaction of NMT1 and EGR2.

Figure 5

  • A
    In vitro pull‐down assay showing the interaction of NMT1 and EGR2. Recombinant GST‐NMT1 was incubated with immunoprecipitated MBP‐His‐EGR2 or MBP‐His at 4°C for 2 h. Anti‐GST and anti‐MBP were used to detect NMT1, EGR2, and MBP‐His, respectively.
  • B
    Co‐localization of NMT1 and EGR2 in N. benthamiana leaves. Scale bar: 50 μm.
  • C, D
    Effect of cold stress on the interaction of EGR2 and NMT1 determined by co‐IP assay. Arabidopsis protoplasts expressing HF/NMT1‐Myc and HF‐EGR2/NMT1‐Myc were treated with or without 4°C for 2 h. Total proteins were immunoprecipitated by anti‐HA agarose beads. Anti‐Myc and anti‐HA were used to detect NMT1‐Myc and HF‐EGR2, respectively. In (C), relative band intensity is shown below the co‐IP blot. In (D), each bar represents the mean ± SEM of three biological experiments (**P < 0.01, two‐tailed t‐test).
  • E
    In vitro pull‐down assay showing decreased interaction intensity of EGR2 (4°C) and NMT1 compared with that of EGR2 (22°C) and NMT1. Total proteins prepared from 2‐week‐old EGR2‐Myc plants grown at 4°C for 0 and 6 h were immunoprecipitated by anti‐Myc agarose beads and incubated with GST‐NMT1 in pull‐down buffer for 2 h at 4°C. Anti‐Myc and anti‐GST antibodies were used to detect EGR2 and NMT1 proteins. Relative band intensity is shown below the output blot.
  • F
    Expression of NMT1 in NMT1‐Myc‐overexpressing plants. EF1α was used as a loading control.
  • G, H
    Freezing phenotype (G) and survival rate (H) of NMT1‐Myc‐overexpressing transgenic plants. Two‐week‐old Super:NMT1‐Myc plants were treated at −5°C (NA) or −8°C for 0.5 h (CA). In (H), each bar represents the mean ± SEM of three biological experiments, and asterisks indicate significant differences compared with the wild type for the same treatment (*P < 0.05, two‐tailed t‐test).
  • I, J
    OST1 kinase activity in NMT1‐Myc‐overexpressing plants under cold stress. Top, autoradiograph; bottom, CBB staining. Representative pictures are shown in (I), and the relative kinase activity is shown in (J). In (I), the ratio of band intensity of OST1 to RuBisCO in wild type with cold treatment for 2 h was set to 1.00. In (J), each bar represents the mean ± SEM of three independent experiments (**P < 0.01, two‐tailed t‐test).

Source data are available online for this figure.