human ubch5b  (Boston Biochem)

Journal: Nature Communications

Article Title: The RING-finger ubiquitin E3 ligase TaPIR1 targets TaHRP1 for degradation to suppress chloroplast function

doi: 10.1038/s41467-024-51249-1

Figure Lengend Snippet: a Detection of TaPIR1–TaHRP1 interaction using the yeast two-hybrid assay. Yeast transformants with marked constructs were grown on SD medium lacking LW (-Leu/-Trp) and those with LacZ activities were grown on SD-LWHA (-Leu/-Trp/-His/-Ade) supplemented with X-α-gal. b Detection of TaPIR1–TaHRP1 interaction in the nucleus of N . benthamiana leaves expressing the corresponding constructs based on the BIFC assay. A NLS fused with a red fluorescent protein (RFP) was used as a nucleus marker. Bar = 20 µm. Similar results are obtained from two independent biological experiments. c Verification of TaPIR1–TaHRP1 interaction using GST pull-down assay. Western blots of the recombinant protein mixture and proteins eluted from GST beads were detected with anti-His or anti-GST antibodies. Similar results are obtained from three independent biological experiments. d Confirmation of the TaPIR1–TaHRP1 interaction via Co-IP assays. Total proteins from N . benthamiana leaves expressing the labeled constructs and proteins copurified from GFP beads were detected via western blotting with anti-GFP and anti-HA antibodies. Similar results are obtained from two independent biological experiments. e Ubiquitination of TaHRP1 by TaPIR1 in vitro. Ubiquitinated TaHRP1, GST-tagged TaHRP1, and His-tagged TaPIR1 were separated and detected via western blotting with anti-ubiquitin, anti-GST, and anti-His antibodies, respectively. Similar results are obtained from two independent biological experiments. f Confirmation of TaHRP1 ubiquitination by TaPIR1 in protoplasts prepared from tapir1-AB , TaPIR1-overexpression, tapir1-AB -expressing TaPIR1 and Fielder plants. Ubiquitinated proteins copurified from the total proteins were detected with an anti-ubiquitin antibody. Similar results are obtained from two independent biological experiments. Source data are provided as a Source Data file.

Article Snippet: E2-622-100), 2 μg of ubiquitin (R&D SYSTEMS, cat.no.

Techniques: Y2H Assay, Construct, Expressing, Bimolecular Fluorescence Complementation Assay, Marker, Pull Down Assay, Western Blot, Recombinant, Co-Immunoprecipitation Assay, Labeling, In Vitro, Over Expression

Journal: Nature Communications

Article Title: The RING-finger ubiquitin E3 ligase TaPIR1 targets TaHRP1 for degradation to suppress chloroplast function

doi: 10.1038/s41467-024-51249-1

Figure Lengend Snippet: The RING-finger ubiquitin E3 ligase TaPIR1 is translocated into the plant nucleus, where it interacts with and ubiquitinates the atypical transcription factor TaHRP1. TaPIR1–TaHRP1 interaction and modification promote TaHRP1 degradation and interfere with TaHRP1-induced PhANGs expression, resulting in attenuated chloroplast-derived ROS accumulation. In tapir1 -AB plants, TaHRP1 accumulation activates PhANGs expression and induces ROS accumulation, which contributes to resistance to Pst in wheat.

Article Snippet: E2-622-100), 2 μg of ubiquitin (R&D SYSTEMS, cat.no.

Techniques: Modification, Expressing, Derivative Assay