Journal: PLoS ONE
Article Title: An Aquaporin 3-Notch1 Axis in Keratinocyte Differentiation and Inflammation
doi: 10.1371/journal.pone.0080179
Figure Lengend Snippet: (A) HKCs reverse transfected with siRNAs against CSL or scrambled control were analyzed 1 week later (in differentiating condition) by qRT-PCR and immunoblot of the indicated genes and proteins. mRNA levels were normalized to reference ribosomal gene RPLP0, and presented as mean fold-change over control ± S.E.M. n=3. ** p< 0.01, *** p < 0.001, **** p<0.0001, Hes1, Keratin 10 and Involucrin were used as indicators of endogenous Notch activity, γ-tubulin served as loading control. (B) Differentiating HKCs were treated with DAPT (10μM) or DMSO control for 72 hours followed by qRT-PCR and immunoblot analysis of the indicated genes and proteins. The analysis was done as in (A). **** p<0.0001, n=3. (C) Proliferating HKCs were infected with retroviruses expressing activated Notch1 or GFP followed, 72 hours later, by qRT-PCR and immunoblot analysis of AQP3. Hey1 was used as an indicator of endogenous Notch activity. The analysis was done as in (A). * p<0.05, *** p<0.001, n=3. (D) Chromatin immuno-precipitation (ChIP) analysis of CSL binding to the regulatory region of the AQP3 gene. Left panel: schematic representation of the AQP3 gene locus based on ChIP-seq information on different chromatin states as provided by the ENCODE project. The location of CSL binding site in the AQP3 gene was analyzed using Mat-inspector software. Right panel: HKCs were processed for ChIP analysis using CSL antibody or non immune IgG. Results are expressed as mean fold of enrichment ± S.E.M. n=2, for each indicated binding site, after immunoprecipitation with CSL antibody or non immune IgG, relative to enrichment for the negative control region.
Article Snippet: The processed chromatin was used for ChIP assays using the ChIP assay kit (Millipore) with CSL antibody (5313) from Cell Signaling.
Techniques: Transfection, Control, Quantitative RT-PCR, Western Blot, Activity Assay, Infection, Expressing, Chromatin Immunoprecipitation, Binding Assay, ChIP-sequencing, Software, Immunoprecipitation, Negative Control
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