(a) HMEECs were treated with curcumin (20 μM) for 1 h, followed by stimulation with NTHi for various time intervals as indicated in the figure. Phospho-IKKβ, total IKKβ protein levels were visualized by western blot. (b) HMEECs were transfected with Mock or IKKβ–CA plasmid. Cells were treated with curcumin (20 μM) for 1 h, and CXCL5 mRNA expression was measured. (c) HMEECs were treated with curcumin (20 μM) for 1 h, followed by stimulation with NTHi for various time intervals as indicated in the figure. Phospho-p38, total p38 protein levels were visualized by western blot. (d,e) HMEECs were pre-treated with curcumin (20 μM) for 1 h, followed by NTHi stimulation for (d) 30 min, (e) 1 h. (d) Phospho-p38, total p38 protein levels (Rhodamine stain), (e) p65 translocation (FITC stain) was visualized by immunofluorescence. DAPI, nuclear stain. Magnification: 400x. (f) HMEECs were transfected with NF-κB luciferase vector. Cells were pre-treated with curcumin (20 μM) for 1 h, followed by stimulated with NTHi for 5 h. NF-κB promoter activity was measured by luciferase assay. Data are mean ± s.d. (n = 3). (b,f) *p < 0.05, t-test. Displayed immunoblots are cropped images from full-length blots, presented in Supplementary Fig. S3. Data are representative of three or more independent experiments.