Journal: Molecular Cell

Article Title: Ubiquiton—An inducible, linkage-specific polyubiquitylation tool

doi: 10.1016/j.molcel.2023.11.016

Figure Lengend Snippet:

Article Snippet: Cycloheximide (CHX) , Merck KgaA , Cat# C7698.

Techniques: Recombinant, Ubiquitin Proteomics, Protease Inhibitor, Modification, Staining, Western Blot, Bicinchoninic Acid Protein Assay, Mass Spectrometry, Knock-Out, Software, Protein Purification

Journal: Nature Communications

Article Title: The ubiquitin ligase Pellino1 targets STAT3 to regulate macrophage-mediated inflammation and tumor development

doi: 10.1038/s41467-025-56440-6

Figure Lengend Snippet: a HEK293T cells were co-transfected with Myc-tagged Pellino1 WT (full-length; FL) or Myc-tagged Pellino1 with a C-terminal deletion (ΔC), Flag-STAT3, and HA-Ub expression plasmids. At 48 h after transfection, cells were treated with MG132 for 5 h, followed by an additional treatment with 1 µg/mL LPS for 30 min. Immunoprecipitation was performed using an anti-Ub antibody. Samples were analyzed by immunoblotting with an anti-STAT3 antibody. The graphic representation depicts Myc-Pellino1 FL and Pellino1 truncation mutants (ΔC), where FHA represents the FHA domain and RING signifies the RING-like domain. b In vitro ubiquitination assay of GST-STAT3 was performed using recombinant E1 (UBE1), recombinant E2 (Ubc13), HA-Ub, and His-Pellino1. Mixtures were incubated at 37 °C in an assay buffer containing ATP for 2 h. Immunoblot analysis was performed using anti-STAT3 and anti-Ub antibodies. c HEK293T cells were co-transfected with Myc-Pellino1, Flag-STAT3, along with HA-Ub WT, HA-K48 Ub, and HA-K63 Ub. At 48 h post-transfection, cells were treated with MG132 for 5 h and subsequently stimulated with 1 μg/mL LPS for 30 min. Cell lysates were immunoprecipitated with an anti-Ub antibody and immunoblotted with an anti-STAT3 antibody. d HEK293T cells were co-transfected with Myc-Pellino1, Flag-STAT3, along with HA-Ub WT, HA-K48R Ub, and HA-K63R Ub. Experimental procedures were identical to those described in ( c ). e Intestinal macrophages were isolated from WT and Pellino1-mKO male mice of normal and acute 1.5% DSS groups. Cells were lysed and immunoprecipitated using an anti-Ub antibody. Immunoprecipitated samples were then analyzed by immunoblotting with an anti-STAT3. f WT and Pellino1-mKO BMDMs were treated with 100 ng/mL LPS for 3 h and then lysed. Cell lysates were immunoprecipitated with an anti-p-STAT3 antibody and immunoblotted with an anti-Ub antibody. Source data are provided as a Source Data file.

Article Snippet: LPS (L5293) and cycloheximide (CHX, C7698) were obtained from Sigma-Aldrich, and MG132 (M-1157) was sourced from AG Scientific.

Techniques: Transfection, Expressing, Immunoprecipitation, Western Blot, In Vitro, Ubiquitin Assay, Recombinant, Incubation, Isolation