Figure 1.

Ischemia activates the canonical and non-canonical NF-κB pathways in HUVEC. Cells were exposed to either normoxia (21% O₂) or ischemia (2% oxygen and nutrient starvation) for 24 h followed by isolation of cytoplasmic and nuclear fractions. (a) The cytoplasmic fractions were analyzed for expression of IκBα by Western blots using specific antibody, and (b) signals were quantified using densitometry by measuring the ratio of band intensity to total protein in the corresponding lanes of the Ponceau S-stained blots. (c) Western blots of the nuclear fractions were probed with anti-p65/RelA antibody and (d) signal quantified by ratio of p65 band intensity to total protein in the lanes, (e) Western blot for non-canonical pathway using an anti-RelB antibody and (f) quantitation of the signal intensity. The total protein from the respective lanes of Ponceau S-stained blots was used. Unpaired t-test with Welch’s correction and two-tailed distribution was used for statistical calculations. Asterisks denote P < 0.05, and n= 6 to 12 in each group.