Figure 5

WB and Co-IP of β-catenin and p65. (A) Wnt3a interfered with NF-κB p50/p65-mediated inflammatory response as suggested by WB analysis of untreated Wnt3a (20 ng/mL) and/or LPS (5 μg/mL)-treated cells at T0, T30′, T1h, and T2h. The quantification of protein expression levels was performed using the image processing software ImageJ. Data were reported as ratio within target protein and relative lamin B housekeeping protein expression. Statistical significance was calculated by Student’s t-test comparing to control: p value ≤0.05*, p value ≤0.01**. (B) Co-IP assay confirmed a nuclear interplay between Wnt signaling and LPS NF-кB pathway, as previously reported [67]. Immunoaffinity purification was carried out using mouse anti-rat NF-κB p65 and mouse anti rat β-catenin antibodies pre-immobilized onto Protein A Sepharose.