Fig. 8: Pellino1-mediated ubiquitination enhances STAT3 activity by increasing the stability of p-STAT3.

a (Left) Immunoblotting of STAT3 and p-STAT3 (Y705) in WT and Pellino1-mKO BMDMs treated with 12.5 μg/mL CHX for the time period indicated in the figure. Cells were stimulated with 100 ng/mL LPS for 3 h before CHX treatment. (Right) Levels of phosphorylated STAT3 were quantified by densitometric analysis of immunoblots using ImageJ (n = 4). b HEK293T cells were transfected with Myc or Myc-Pellino1. After 48 h of co-transfection, cells were treated with 100 μg/mL CHX for the time period indicated in the figure. Cells were stimulated with 1 μg/mL LPS for 30 min before CHX treatment. c HEK293T cells were co-transfected with Myc-Pellino1, HA-Ub WT, HA-K63 Ub, and HA-K63R Ub. Experimental procedures were identical to those described in (b). d WT and Pellino1-mKO BMDMs were fractionated after 3 h of treatment with 100 ng/mL LPS. Immunoblot analysis was performed to assess p-STAT3 (Y705) protein levels in the cytoplasm and nucleus. CF cytosolic fraction, NF nuclear fraction. e ChIP-qRT-PCR analysis was performed using anti-STAT3 or control IgG with WT and Pellino1-mKO BMDMs as shown in the figure. BMDMs were stimulated with 100 ng/mL LPS for 6 h. ChIP data are presented as relative enrichment normalized to input in WT or Pellino1-mKO BMDMs (n = 3). f MMP9, BCL2, and IL6 mRNA levels in WT and Pellino1 deficient BMDMs (n = 3) were quantified with qRT-PCR and normalized against GAPDH expression. g MMP9, BCL2, IL6, and VEGF mRNA levels in intestinal macrophages from WT and Pellino1-mKO male mice of normal, acute 1.5% DSS, and AOM/DSS groups (n = 4) were quantified with qRT-PCR and normalized against GAPDH expression. Data were represented as mean ± SD in (a, e–g). All statistical comparisons were made using a two-tailed Student’s t-test. Source data are provided as a Source Data file.