Figure 3
From: A TNF-induced gene expression program under oscillatory NF-κB control
Validation of expression profiles and NF-κB dependence. (a) Early gene profiles. HeLatTA/FLAG-IκBα Mut cells were plated in parallel in the absence or presence of Dox (2 μg/ml) and stimulated with rhTNFα. Changes in mRNA abundance (normalized by 18S) determined by Q-RT- PCR from total RNA. For each of the indicated mRNA transcripts, values are expressed as fold change relative to unstimulated cells and plotted on a logarithmic scale. +/-Dox, data obtained from cells cultured with or without Dox. (b) Late gene profiles. Experiment and data analysis are as in Figure 3a. (c) ChIP for NF-κB subunit binding to Early Gene promoters. ChIP was performed on control or TNFα-stimulated (30 min, 20 ng/ml) HeLa cells using the antibodies indicated at left. Shown is an ethidium-bromide stained agarose gel of the PCR products performed under linear amplification conditions. The target gene is indicated at the bottom. NC, negative control reaction (no template is added to the PCR reaction); PC, positive control reaction (25 ng of genomic DNA is used as template in PCR). (d) ChIP for NF-κB subunit binding to Late Gene promoters. ChIP was performed on HeLa cells stimulated as in Figure 3c.