Journal: Plant Biotechnology Journal

Article Title: AaSIZ1 ‐Mediated SUMOylation of AaMYB31 Positively Regulates Freezing Tolerance in Actinidia arguta

doi: 10.1111/pbi.70620

Figure Lengend Snippet: AaSIZ1‐mediated SUMOylation of AaMYB31s. (A) In vitro SUMOylation of AaMYB31s. SUMOylated GST‐AaMYB31.1 and GST‐AaMYB31.2 were detected with anti‐GST and anti‐SUMO1 antibodies. (B) In vivo SUMOylation of AaMYB31s in Actinidia arguta protoplasts. AaMYB31.1‐Myc or AaMYB31.2‐Myc was co‐expressed with FLAG‐SUMO1 GG or FLAG‐SUMO1 AA in Actinidia arguta protoplasts. Myc‐AaMYB31.1 and Myc‐AaMYB31.2 were immunoprecipitated with an anti‐Myc antibody, and the immunoprecipitated proteins were detected with anti‐Myc and anti‐FLAG antibodies. (C) In vivo SUMOylation of AaMYB31s in N. benthamiana . AaMYB31.1‐GFP or AaMYB31.2‐GFP was co‐expressed with FLAG‐SUMO1 GG or FLAG‐SUMO1 AA in N. benthamiana leaves. AaMYB31.1‐GFP and AaMYB31.2‐GFP were immunoprecipitated with an anti‐GFP antibody, and the immunoprecipitated proteins were detected with anti‐GFP and anti‐FLAG antibodies. (D) In planta SUMOylation of AaMYB31s. Total proteins were extracted from freshly harvested leaves of WT Actinidia chinensis , 35S::AaMYB31.1‐GFP , and 35S::AaMYB31.2‐GFP transgenic plants, and the AaMYB31.1‐GFP and AaMYB31.2‐GFP were immunoprecipitated with an anti‐GFP antibody. The immunoprecipitated proteins were detected with anti‐GFP and anti‐SUMO1 antibodies. (E) In vivo SUMOylation of AaMYB31.1‐GFP, AaMYB31.2‐GFP, AaMYB31.1 K76R ‐GFP, and AaMYB31.1 K76R ‐GFP in N. benthamiana leaves. AaMYB31.1‐GFP, AaMYB31.1 K76R ‐GFP, AaMYB31.2‐GFP, or AaMYB31.2 K76R ‐GFP was co‐expressed with FLAG‐SUMO1 GG or FLAG‐SUMO1 AA in N. benthamiana leaves. AaMYB31.1‐GFP and AaMYB31.2‐GFP were co‐expressed with FLAG‐SUMO1 AA as a negative control. AaMYB31.1‐GFP or AaMYB31.2‐GFP was immunoprecipitated with an anti‐GFP antibody, and the immunoprecipitated proteins were detected with anti‐GFP and anti‐FLAG antibodies. (F) In planta SUMOylation of AaMYB31.1‐GFP, AaMYB31.2‐GFP, AaMYB31.1 K76R ‐GFP, and AaMYB31.1 K76R ‐GFP. Total proteins were extracted from freshly harvested leaves of 35S::AaMYB31.1‐GFP , 35S::AaMYB31.2‐GFP , 35S::AaMYB31.1 K76R ‐GFP , and 35S::AaMYB31.1 K76R ‐GFP transgenic plants, and the GFP‐fusion proteins were immunoprecipitated with an anti‐GFP antibody. The immunoprecipitated proteins were detected with anti‐GFP and anti‐SUMO1 antibodies. (G) AaSIZ1 SUMOylated AaMYB31s protein in vitro. SUMOylated GST‐AaMYB31.1 and GST‐AaMYB31.2 were detected with anti‐GST and anti‐SUMO1 antibodies. (H) In vivo AaSIZ1 SUMOylation of AaMYB31s in N. benthamiana . AaMYB31.1‐GFP or AaMYB31.2‐GFP and AaSIZ1 were co‐expressed with or without FLAG‐SUMO1 in N. benthamiana leaves. AaMYB31.1‐GFP and AaMYB31.2‐GFP were immunoprecipitated with an anti‐GFP antibody, and the immunoprecipitated proteins were detected with anti‐GFP and anti‐FLAG antibodies. (I) The SUMOylated of AaMYB31s in the 35S::AaSIZ1‐GFP transgenic plants. Total proteins were extracted from freshly harvested leaves of WT Actinidia chinensis and 3 5S::AaSIZ1‐GFP transgenic plants and incubated with GST‐AaMYB31.1 or GST‐AaMYB31.2. SUMOylated GST‐AaMYB31.1 and GST‐AaMYB31.2 were detected with anti‐GST antibody. (J) AaSIZ1 mediates the SUMOylation of AaMYB31s in Actinidia arguta protoplasts. Myc‐AaMYB31.1 or Myc‐AaMYB31.2 was co‐expressed with FLAG‐SUMO1 in Actinidia arguta protoplasts. Myc‐AaMYB31.1 and Myc‐AaMYB31.2 were immunoprecipitated with an anti‐Myc antibody, and the immunoprecipitated proteins were detected with anti‐Myc and anti‐FLAG antibodies.

Article Snippet: Then the protein was immunoprecipitated with anti‐GFP (AE012, ABclonal; dilution 1:2000) and anti‐Myc (AE010, ABclonal; dilution 1:2000) antibodies, and the immunoprecipitated proteins were detected with anti‐Myc, anti‐FLAG and anti‐GFP antibodies.

Techniques: In Vitro, In Vivo, Immunoprecipitation, Transgenic Assay, Negative Control, Incubation

Journal: Plant Biotechnology Journal

Article Title: AaSIZ1 ‐Mediated SUMOylation of AaMYB31 Positively Regulates Freezing Tolerance in Actinidia arguta

doi: 10.1111/pbi.70620

Figure Lengend Snippet: AaSIZ1‐mediated SUMOylation of AaMYB31s enhances their protein stability. (A, B) The protein degradation of AaMYB31s in the AaMYB31.1‐GFP and AaMYB31.1 K76R ‐GFP , or AaMYB31.2‐GFP and AaMYB31.1 K76R ‐GFP transgenic plants. Total proteins were extracted from freshly harvested leaves of AaMYB31.1‐GFP and AaMYB31.1 K76R ‐GFP , or AaMYB31.2‐GFP and AaMYB31.1 K76R ‐GFP transgenic plants. AaMYB31.1‐GFP, AaMYB31.1 K76R ‐GFP, AaMYB31.2‐GFP, and AaMYB31.2 K76R ‐GFP were detected with anti‐GFP antibody. ACTIN was used as an internal control. (C, D) In vivo protein degradation of AaMYB31s in N. benthamiana . 35S::GFP or 35S::AaSIZ1‐GFP was co‐expressed with Myc‐AaMYB31s and HA‐RFP in N. benthamiana leaves, and the total protein was extracted. These proteins were treated with DMSO (−MG132) or 50 μM MG132 for the indicated periods of time and sampled simultaneously to detect. Myc‐AaMYB31.1 and Myc‐AaMYB31.2 were detected with anti‐Myc antibody. HA‐RFP was used as an internal control. (E, F) The protein degradation of AaMYB31s in the 35S::AaSIZ1‐GFP transgenic plants. Total proteins were extracted from freshly harvested leaves of WT Actinidia chinensis and 3 5S::AaSIZ1‐GFP transgenic plants and incubated with GST‐AaMYB31.1 or GST‐AaMYB31.2. These proteins were treated with DMSO (−MG132) or 50 μM MG132 for the indicated periods of time and sampled simultaneously to detect. GST‐AaMYB31.1 and GST‐AaMYB31.2 were detected with anti‐GST antibody. ACTIN was used as an internal control.

Article Snippet: Then the protein was immunoprecipitated with anti‐GFP (AE012, ABclonal; dilution 1:2000) and anti‐Myc (AE010, ABclonal; dilution 1:2000) antibodies, and the immunoprecipitated proteins were detected with anti‐Myc, anti‐FLAG and anti‐GFP antibodies.

Techniques: Transgenic Assay, Control, In Vivo, Incubation

Journal: eLife

Article Title: BLOS1 mediates kinesin switch during endosomal recycling of LDL receptor

doi: 10.7554/eLife.58069

Figure Lengend Snippet:

Article Snippet: Antibody , Mouse monoclonal anit-Myc tag antibody (clone Myc3) , MBL , Cat#M192-3S; RRID: AB_11161202 , IF (1:500), WB (1:2000).

Techniques: Derivative Assay, Isolation, Recombinant, Plasmid Preparation, Sequencing, Mutagenesis, shRNA, cDNA Synthesis, Cloning, Software