ubiquitin e3 enzyme parkin  (Boston Biochem)

Journal: iScience

Article Title: Trichinella spiralis inhibits myoblast differentiation by targeting SQSTM1/p62 with a secreted E3 ubiquitin ligase

doi: 10.1016/j.isci.2024.109102

Figure Lengend Snippet: Trichinella spiralis -secreted Ts -RNF has E3 ubiquitin ligase activity (A) In vitro ubiquitination assay showing the E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligase activity of muscle larvae excretory-secretory products (ML-ES); ML-ES was incubated with HA-Ub, E1 (UBE1), HisS5a, and ATP buffer at 37°C for 2 h in the presence or absence of E2 (UbcH5A) or E3 (Parkin). After the termination, the reaction mixture was subjected to SDS-PAGE and immunoblotting assay with an anti-HA antibody. (B) In vitro ubiquitination assay showing the E3 ubiquitin ligase activity of Ts -RNF. The Ts -RNF protein was incubated with HA-Ub, E1 (UBE1), E2 (UbcH5A), HisS5a, and ATP buffer in the presence or absence of the E3 (Parkin) protein at 37°C for 2 h. After the termination, the reaction mixture was subjected to SDS-PAGE and immunoblotting assay with an anti-HA antibody. (C) Ts -RNF-specific antibody was made and its specificity was assessed by immunoblotting against muscle larvae lysate ( Ts -ML), ML-ES, and uninfected skeletal muscle tissue lysate (SMT). (D) Expression analysis of Ts -RNF at different developmental stages (n = 3). ML, muscle larvae; AD, adults; NBL, newborn larvae. Data are shown as the means ± SEMs from three independent experiments. Statistical analysis was performed using one-way ANOVA test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; ns, not significant. (E) Confocal microscopy analysis demonstrating colocalization of ubiquitin (green), Ts -RNF (red), and Hoechst (blue) in the skeletal muscle of mice infected with T. spiralis . For the Ub negative control, PBS was used to replace the primary antibody, and for the Ts -RNF negative control, the primary antibody was replaced with preimmune serum. Representative single optical sections and merged images are shown. Scale bars: 50 μm.

Article Snippet: Ubiquitin E3 Enzyme Parkin , Boston Biochem, USA , Cat#E3-160.

Techniques: Ubiquitin Proteomics, Activity Assay, In Vitro, Incubation, SDS Page, Western Blot, Expressing, Confocal Microscopy, Infection, Negative Control

Journal: iScience

Article Title: Trichinella spiralis inhibits myoblast differentiation by targeting SQSTM1/p62 with a secreted E3 ubiquitin ligase

doi: 10.1016/j.isci.2024.109102

Figure Lengend Snippet: Ts -RNF interacts with SQSTM1/p62 K422 via K63 linker modification (A) The Flag- Ts -RNF and Myc-p62, △13-165, △187-297, and △334-434 expression plasmids were cotransfected into HEK293T cells, and co-IP experiments were performed to determine the interaction between Ts -RNF and p62 using anti-FLAG beads followed by immunoblotting. (B) The Flag- Ts -RNF and Myc-p62, 422K/R, and 437K/R expression plasmids were cotransfected into HEK293T cells, and co-IP experiments were performed with anti-FLAG beads followed by immunoblotting. (C) The Flag- Ts -RNF, Myc-p62, and HA-Ub, K6, K11, K27, K29, K33, K48, and K63 expression plasmids were cotransfected into HEK293T cells, and co-IP experiments were performed to determine the ubiquitination linkage antibodies between Ts -RNF and p62 by anti-Myc beads followed by immunoblotting. (D) The in vitro ubiquitination assay showing the interaction between Ts -RNF and p62. The Ts -RNF protein was incubated with HA-Ub, E1 (UBE1), E2 (UbcH5A), E3 (Parkin), and ATP buffer in the presence or absence of p62 protein at 37°C for 2 h. Parkin auto-ubiquitination reaction was utilized as a positive control. After the termination, the reaction mixture was subjected to SDS-PAGE and immunoblotting assay with an anti-HA antibody. (E) In vitro ubiquitination assay showing K63-linked ubiquitin chains between Ts -RNF and p62. Ts -RNF and p62 protein were incubated with His-Ub (WT, K48, K63R), E1 (UBE1), E2 (UbcH5A), E3 (Parkin), and ATP buffer at 37°C for 2 h. Parkin auto-ubiquitination reaction was utilized as a positive control. After the termination, the reaction mixture was subjected to SDS-PAGE and immunoblotting assay with an anti-His antibody.

Article Snippet: Ubiquitin E3 Enzyme Parkin , Boston Biochem, USA , Cat#E3-160.

Techniques: Modification, Expressing, Co-Immunoprecipitation Assay, Western Blot, Ubiquitin Proteomics, In Vitro, Incubation, Positive Control, SDS Page

Journal: iScience

Article Title: Trichinella spiralis inhibits myoblast differentiation by targeting SQSTM1/p62 with a secreted E3 ubiquitin ligase

doi: 10.1016/j.isci.2024.109102

Figure Lengend Snippet:

Article Snippet: Ubiquitin E3 Enzyme Parkin , Boston Biochem, USA , Cat#E3-160.

Techniques: Recombinant, Ubiquitin Proteomics, Conjugation Assay, Transfection, Plasmid Preparation, Software

Journal: bioRxiv

Article Title: Development and characterization of phospho-ubiquitin antibodies to monitor PINK1-PRKN signaling in cells and tissue

doi: 10.1101/2024.01.15.575715

Figure Lengend Snippet: Assessing the top p-S65-Ub antibodies in ELISA using recombinant protein. ( A ) Sandwich ELISA results for the detection of recombinant K48 (solid line) and K63 (dash line) p-S65-Ub tetramers (Ub4) that were serially diluted to the range of 1–1,000,000 pg/ml. ( B ) Direct ELISA results for the detection of p-S65-PRKN and nonphosphorylated PRKN recombinant proteins that were serially diluted to the range of 1–1,000,000 pg/ml. N = 2. Data are shown as mean with SD.

Article Snippet: Recombinant p-S65-PRKN (Boston Biochem, E3–166), non-phosphorylated PRKN (Boston Biochem, E3–160) and p-S65-Ub monomer (Boston Biochem, U102) were initially dissolved in RIPA buffer (stock concentration of 1 μg/μl), aliquoted in 1 μl aliquots, flash-frozen in liquid nitrogen and stored at −80°C.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Sandwich ELISA, Direct ELISA