ube2i primary antibody  (Proteintech)

Journal: The international journal of biochemistry & cell biology

Article Title: PIAS1 S510G variant acts as a genetic modifier of spinocerebellar ataxia type 3 by selectively impairing mutant ataxin-3 proteostasis.

doi: 10.1016/j.biocel.2024.106662

Figure Lengend Snippet: Fig. 5. Biochemical analysis of the interaction between ATXN3, PIAS1, and UBC9, and PIAS1-mediated ATXN3 SUMOylation in vitro. (A) Diagram showing the ternary complex formed by PIAS1, ATXN3, and UBC9 proteins. (B) Analysis of PIAS1 and ATXN3 interaction by incubating purified recombinant PIAS1 proteins with ATXN3 proteins produced by in vitro transcription/translation. The samples were then subjected to immunoprecipitation with ATXN3 antibody followed by Western blot analysis using PIAS1 antibody. (C) Analysis of PIAS1 and UBC9 interaction by GST pull-down assay. GST-Ubc9 was incubated with PIAS1-WT or PIAS1-S510G protein and the pull-down samples were probed using PIAS1 antibody. The level of PIAS1-WT interacting with GST-UBC9 was set at 100 %. GST was included as a negative control. (D) Analysis of PIAS1 and UBC9 interaction in the presence of ATXN3–28Q or ATXN3–84Q as described above. Data from independent biological replicates are shown in the bar graphs and were analyzed using Student’s t-test (N=3). (E) In vitro SUMOylation assay to evaluate the effect of PIAS1-S510G on the SUMO conjugation of ATXN3–28Q and ATXN3–84Q. ATXN3–28Q and ATXN3–84Q were captured by ATXN3 antibody with protein G agarose beads.

Article Snippet: DNA fragments of UBC9, PIAS1-WT and PIAS1-S510G were cloned into pTriEx-Rac1–2 G (Addgene plasmid # 66110) to create pTriEx-CFPUBC9, pTriEx-PIAS1WT -YFP and pTriEx-PIAS1S510G-YFP for FRET analysis.

Techniques: In Vitro, Purification, Recombinant, Produced, Immunoprecipitation, Western Blot, Pull Down Assay, Incubation, Negative Control, Conjugation Assay

Journal: The international journal of biochemistry & cell biology

Article Title: PIAS1 S510G variant acts as a genetic modifier of spinocerebellar ataxia type 3 by selectively impairing mutant ataxin-3 proteostasis.

doi: 10.1016/j.biocel.2024.106662

Figure Lengend Snippet: Fig. 6. Analysis of the protein-protein interaction among PIAS1, UBC9, and ATXN3 in cells by FRET assay. (A) HeLa cells expressing CFP-UBC9 along with PIAS1- WT-YFP or PIAS1-S510G-YFP were examined under a fluorescence microscope. Scale bar = 10 μm. (B) Quantification of FRET efficiency in PIAS1-WT (n=40) and PIAS1-S510G (n=33) from three independent biological replicates. Data are presented as a box plot. Statistical significance was determined using Student’s t-test. (C) Representative images of HeLa cells expressing CFP-UBC9 with PIAS1-WT-YFP or PIAS1-S510G-YFP in the presence of ATXN3–28Q and ATXN3–84Q. (D) Quan tification of FRET efficiency in C. ATXN3–28Q with PIAS1-WT (n=42) or PIAS1-S510G (n=42) and ATXN3–84Q with PIAS1-WT (n=42) or PIAS1-S510G (n=31) were analyzed from three independent biological replicates. (E) Protein lysates of cells in C were collected for Western blot analysis.

Article Snippet: DNA fragments of UBC9, PIAS1-WT and PIAS1-S510G were cloned into pTriEx-Rac1–2 G (Addgene plasmid # 66110) to create pTriEx-CFPUBC9, pTriEx-PIAS1WT -YFP and pTriEx-PIAS1S510G-YFP for FRET analysis.

Techniques: Expressing, Fluorescence, Microscopy, Western Blot