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Fig. 4 Pinostrobin downregulates nuclear accumulation of NF-κB. (A) RAW 264.7 macrophages were pretreated with pinostrobin (0–20 μM) 2 h before treatment with 300 ng/mL LPS for 30 min. Nuclear proteins were collected and used for western blotting. Nucleolin was used for normalizing p50 and p65 expression. The relative density was calculated using ImageJ software. (B) In a parallel experiment, p65 was immunostained with anti-p65 antibody (Ab) and detected using secondary Ab conjugated with Alexa Fluor 647 (red) followed by DAPI (blue) staining for nuclei (scale bar = 100 µm). The fluorescence was analyzed using a CELENA S Digital Imaging System. ###, p < 0.001 vs. untreated cells; ***, p < 0.001 vs. LPS-treated cells.