Fig. 2

ECPCM does not affect TNFα-dependent activation of NF-kB or nuclear translocation of p65 in HAEC. a HAEC were treated for 30 min with collection medium or with TNFα in collection medium or ECPCM. Western blot analysis was performed using antibodies for β-actin and IkBα. TNFα induced IkBα degradation, and therefore NF-kB pathway activation, when collection medium or ECPCM was used during the treatment. The experiment was repeated at least three times with similar results, and representative blots are shown. Bottom panel, relative densitometric quantification of western blot bands for TNFα-treated HAEC. IκBα bands were normalised to the β-actin loading control band. Data = mean ± SEM; n = 5 per treatment. p-value: * <0.05 compared to collection medium control. b HAEC were treated with or without TNFα in collection medium or ECPCM for 10 min, 30 min, 1 h or 2 h. Immunofluorescence staining was performed with anti-PECAM-1 (red) and anti-p65 (green) antibodies. TNFα induced nuclear translocation of p65 upon treatment in both collection medium and ECPCM. Control: cells incubated in collection medium or ECPCM without TNFα for 2 h. Some nuclei are outlined with white dots to highlight the translocation of p65