Fig. 6: Pellino1 functionally interacts with STAT3 signaling. | Nature Communications

Fig. 6: Pellino1 functionally interacts with STAT3 signaling.

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

Fig. 6

a Immunoblot analysis was performed to assess Pellino1, STAT3, and p-STAT3 (Y705) protein levels in lysates of intestinal macrophages isolated from WT and Pellino1-mKO male mice in normal, acute 1.5% DSS and AOM/DSS groups. Actin was used as a loading control. b Immunoblot analysis was performed to assess Pellino1, STAT3, and p-STAT3 (Y705) protein levels in lysates of BMDMs from WT and Pellino1-mKO mice. BMDMs were stimulated with 100 ng/mL LPS for the indicated time. Actin was used as a loading control. c GST pulldown assay was performed by incubating RAW cell lysates treated with or without 100 ng/mL LPS for 6 h, along with GST or GST-STAT3. d Co-immunization (Co-IP) assays were performed using HEK293T cells transfected with expression vectors for Myc, Myc-tagged Pellino1 (Myc-Pellino1), and Flag-tagged STAT3 (Flag-STAT3). Cellular extracts were immunoprecipitated with an anti-Myc antibody and immunoblotted with an anti-Flag antibody. e GST (black arrow) or GST-STAT3 (blue arrow) protein was incubated with purified His-Pellino1 and subjected to immunoblotting using anti-Pellino1 and anti-GST antibodies. CS: Coomassie brilliant blue staining. f WT and Pellino1-mKO BMDMs were stimulated with 100 ng/mL LPS for 6 h. Immunofluorescence staining of Pellino1 (red), p-STAT3 (green), and DAPI (blue). DAPI was used to stain the nuclei. Scale bar = 10 μm. g GST pulldown assay was performed using purified GST or GST-Pellino1 protein with RAW cell lysates. RAW cells were stimulated with 100 ng/mL LPS for 6 h. h For IP assays, LPS-untreated RAW cells and RAW cells treated with 100 ng/mL LPS for 6 h were lysed. Subsequently, immunoprecipitation was performed using an anti-Pellino1 antibody, followed by immunoblotting with an anti-STAT3 antibody. i Co-IP assays were performed using HEK293T cells transfected with expression vectors for Myc-Pellino1, Flag-STAT3 WT, and Flag-STAT3 dominant negative (Flag-STAT3 DN; Flag-STAT3 Y705F). Cellular extracts were subjected to immunoprecipitation using an anti-Myc antibody and subsequently immunoblotted with anti-Flag and anti-Myc antibodies. Source data are provided as a Source Data file.

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