NFκB activation in K14-HIF-1αDPM keratinocytes is dependent on increased Erk1/2 phosphorylation. Increased nuclear translocation of the NFκB subunit p65 in transgenic (DPM) versus nontransgenic keratinocytes (NTG), normalized to Sp1 (A). Nuclear extracts were analyzed by Western blots. Western blot of lysates from newborn transgenic (DPM) keratinocytes indicate a 2- to 3-fold increase of phosphorylated IKKα, IκBα, p65 phosphorylation at serines 276 and 536, and phospho-ERK1/2, compared with nontransgenic (NTG) keratinocytes; β-tubulin is a loading control (B). Transgenic keratinocytes were pretreated with DMSO (−) or the MEK1/2 inhibitor U0126 (+) for 24 hours and analyzed at baseline (−) or 10 minutes after TPA treatment (+). Western blots of lysates from newborn transgenic keratinocytes show inhibition of phospho-ERK1/2 and p65 Ser²⁷⁶ phosphorylation by U0126 both at baseline and after TPA treatment (C). In contrast, phospho-IκBα, phospho-p65 Ser⁵³⁶, and the level of IκBα protein remain unaffected by ERK1/2 inhibition (C); total ERK1/2 is used as a loading control. The separating line in panel C represents one lane deleted from the same gel. Real-time RT-PCR analysis of TNFα expression from total RNA extracted from transgenic (DPM) primary keratinocytes shows selective inhibition of TNFα expression 24 hours after U0126 (U0) treatment in contrast to PI3 kinase pathway wortmannin (W), or EGFR AG1478 (AG) inhibition, both of which failed to alter transgenic keratinocyte TNFα expression (D). Bars represent mean plus or minus SEM. Results are representative of 3 independent experiments (*P < .05, t test). (E-F) Increased ERK1/2 phosphorylation is also evident in immunohistochemical analysis of transgenic compared with nontransgenic ears (E,F). Bar in panel E represents 100 μm.