Figure 2.

NF-κB p65 translocates to the nucleus following treatment with dynasore. (A) Western blot: RAW cells were treated with 80 μM dynasore or DMSO in HBSS for 90 min. As a positive control for p65 translocation, cells were treated with 15 ng·mL⁻¹ LPS. Cytoplasm and nuclear extracts’ purity were assessed by reprobing using an antibody against nuclear histones. (B) Immunocytochemistry: RAW cells were plated on coverslips, dynasore was added for 2 h and LPS-positive control was treated for 15 min. Cells were immunostained with anti-NF-κB p65 antibody followed by anti-rabbit Alexa-568 conjugated antibody (red). Hoechst 33342 was used for nuclear staining (blue). (C) To quantify the data in section B, 100 cells in each field were counted for NF-κB p65 localization to the cytoplasm, nucleus or both. Note that while in 99% of control cells NF-κB p65 was localized to the cytoplasm, 90% and 99% of dynasore and LPS-treated cells, respectively, had NF-κB p65 localized to the nucleus. However, while LPS-treated cells localized NF-κB p65 exclusively to the nucleus, the majority of dynasore-treated cells exhibited NF-κB p65 localization to both the cytoplasm and nucleus.