Computational protocol: PHF11 expression and cellular distribution is regulated by the Toll-Like Receptor 3 Ligand Polyinosinic:Polycytidylic Acid in HaCaT keratinocytes

Similar protocols

Protocol publication

[…] Full-length PHF11 cDNA was cloned into the plasmid expression vector pEGFP-C1 (Clontech) and was then used as a template to generate the two C-terminal deletion constructs del218 and del165 that terminate at valine residue 218 and alanine residue 165, respectively. Numbering of amino acids is based on NCBI reference sequence NP_001035533.1. Transfection of HEK cells was done as previously described []. Identification of a putative nuclear localization sequence was done using the following online tool: http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi. Western blot analysis confirmed that each recombinant protein was expressed at the predicted molecular weight (data not shown). Images were analysed using ImageJ (http://imagej.nih.gov/ij/). [...] In experiments to visualize IL-8 production, 0.5 mM Monensin sodium salt (Sigma, St. Louis, USA) was added to the culture media and cells returned to 37 °C/5%CO2 for the final 4 h of stimulation by poly(I:C) []. To visualize IL-8, as well as claudin-1 and PHF11, cells were fixed in a 4 % formaldehyde solution for 10 min at room temperature, washed and permeabilized by washing for 3 × 5 min in PBS/0.01 % Triton X100. Cells were then blocked in Image-iT™ FX Signal Enhancer (Molecular Probes, Oregon, USA) for 30 min and washed a further 3 × 5 min in PBS/0.01 % Triton X100. The cells were incubated with the following primary antibodies in blocking buffer (1%BSA, 0.01 % Triton X100, 5 % FCS in PBS) overnight at 4 °C: rabbit anti-Claudin 1 (1:1000, Sigma-Aldrich, SAB4503546), rabbit anti-PHF11 (1:100, ProteinTech Group, 10898-1-AP), mouse anti-CXCL8/IL-8 (10 μg/ml, R&D systems, MAB208). Cells were then washed for 3 × 5 min in PBS/0.01 % Triton X100 and then incubated with a 1:1000 dilution Alexa Flour® 488 goat anti-rabbit IgG (H + L) and/or a 1:1000 dilution of Alexa Flour® 555 goat anti-mouse IgG (H + L) (Molecular probes/Life Technologies™, Austin, TX, USA) in blocking buffer at room temperature for 1 h in the dark. Cells were washed 2 × 5 min in PBS/0.01 % Triton X100, and nuclear DNA was stained using a solution of 1 μg/ml Hoechst 33342 (Molecular probes/Life Technologies™, Austin, TX, USA) for 10 min at room temperature protected from light. After a final 5 min wash in PBS/0.01 % Triton X100, 2 drops of ProLong® Gold antifade reagent was added to the slide and covered with a glass cover slip. Slides were viewed on an Olympus BX43 Microscope fitted with an X-Cite Series 120Q EXFO Halogen Lamp using cellSens standard software or using a TCS-SP5 confocal microscope (Leica Microsystems, Germany). […]

Pipeline specifications

Software tools ImageJ, cellSens
Applications Laser scanning microscopy, Microscopic phenotype analysis
Diseases Dermatitis, Dermatitis, Atopic, Hypersensitivity, Psoriasis, Skin Diseases