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Fig. 4 | Genome Medicine

Fig. 4

From: Functional screen of inflammatory bowel disease genes reveals key epithelial functions

Fig. 4

Impact of increased DUSP16 expression on MAPKs phosphorylation levels. The impact of DUSP16 expression on ERK, p38 and JNK phosphorylation levels was evaluated using a doxycycline inducible HT-29 cell model. A The impact of DUSP16 on steady-state MAPKs phosphorylation levels was measured. Exponentially growing HT-29-pLVX-Tet3G cells stably transduced with a TET3G-inducible expression plasmid (empty vector (EV) or containing the DUSP16 ORF (DUSP16) were stimulated with doxycycline (1 μg/ml) for 24 h, and cell lysates were harvested for the evaluation of the native and phosphorylated states of MAPKs. Cropped western blots showing bands of interest are shown (left) and the level of phosphorylation of each MAPK is summarized in a graphical representation (right). A single representative gel is shown for EEF2 loading control; full blots along with their respective EEF2 are shown in Additional file 2, Fig.S11, panel A. B The impact of DUSP16 on the induction of MAPKs phosphorylation following serum starvation was measured. Using the same models as above, exponentially growing cells were first serum-starved for 24 h (0% FBS) in the presence of the doxycycline inducer (1 μg/ml). The cells were then stimulated with 10 ng/ml TNF-α for different times to induce MAPK phosphorylation and whole cell lysates harvested for the evaluation of the native and phosphorylated states of MAPKs. The level of phosphorylation of each MAPK is summarized in a graphical representation; the y-axis shows phosphorylation levels relative to the control condition (EV in A and EV 0’ in B). All phosphorylation level data is expressed as the ratio of phosphorylated over total MAPK (both corrected for EEF2 loading control) combining the replicates (n = 3). All results are shown as geometric means with SEM. The values for independent replicates are shown (grey lozenges). As an indication, three non-overlapping datapoints from two conditions correspond to the strongest evidence against the hypothesis of no difference for a non-parametric test (P value = 0.1). Under hypothesis of lognormal distribution, the intervals as plotted correspond to 58% confidence interval for the median. Full blots for each replicate and all timepoints, along with EEF2 results are shown in Additional file 2, Fig.S11, panels B-C

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