Journal: Nature Communications
Article Title: WASH maintains NKp46+ ILC3 cells by promoting AHR expression
Figure Lengend Snippet: WASH associates with Arid1a. ( a ) Yeast strain AH109 was co-transfected with Gal4 DNA-binding domain (BD)-fused WASH and Gal4 activating domain (AD)-fused Arid1a. p53 and large T antigen were introduced as a positive control. ( b ) GST-WASH was incubated with LPL lysates, followed by a GST pulldown assay. ( c ) 4 × 10 5 NKp46 + ILC3 cells (pooled from RORγt-GFP reporter mice) were lysed and immunoprecipitated with anti-WASH antibody, followed by immunoblotting with the indicated antibodies. Lysates were treated by DNase I before incubation with antibodies. 4 × 10 5 Arid1a deleted ILC3 cells sorted from Arid1a flox/flox Rorc -Cre mice were also lysed and immunoprecipitated with anti-WASH antibody for co-IP assay as a control. Experiment was repeated for two times. ( d ) Flag-tagged WT and truncated Arid1a (upper panel) were co-transfected with HA-tagged WASH into NK92 cells, followed by immunoprecipitation with antibody against Flag. Immunoprecipitates were immunoblotted with the indicated antibodies (lower panel). ( e ) NK92 cells with or without Arid1a knockdown were subjected to immunoprecipitation with antibody against WASH, followed by immunoblotting with the indicated antibodies. ( f ) Sorted NKp46 + ILC3 cells were subjected to ChIP assay with antibody against Arid1a, followed by PCR analysis with the indicated fragment primers of Ahr . ( g ) Sorted NKp46 + ILC3 cells were subjected to ChIP assay with antibodies against the indicated BAF components, followed by PCR examination with primers specific to Ahr promoter. ( h ) ILC3s were sorted from Arid1a flox/flox and Arid1a flox/flox Rorc -Cre mice, followed by immunoblotting. ( i ) NKp46 + ILC3 cells from Arid1a flox/flox and Arid1a flox/flox Rorc -Cre mice were subjected to nuclear run-on assay, followed by RT-PCR analysis of Ahr . ( j ) Arid1a overexpressing BM cells together with equal numbers of recipient BM cells were transplanted into lethally irradiated recipient mice. Vector or Arid1a overexpressing ILC3 cells were sorted from above reconstituted mice and subjected to nuclear run-on assay, followed by RT-PCR analysis of Ahr . For i and j ), n =5. Data are shown as means±s.d. * P
Article Snippet: Then nuclei were resuspended with DNase I digestion buffer and treated with indicated units of DNase I (Sigma-Aldrich) at 37°C for 5 min. 2 × DNase I stop buffer (20 mM Tris Ph 8.0, 4 mM EDTA, 2 mM EGTA) was added to stop reactions.
Techniques: Transfection, Binding Assay, Positive Control, Incubation, GST Pulldown Assay, Mouse Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Nuclear Run-on Assay, Reverse Transcription Polymerase Chain Reaction, Irradiation, Plasmid Preparation