Molecule known to slow inflammation linked to scleroderma, could be treatment target umich NatureComms
. The products were amplified using SYBR Green PCR Master Mix on an Applied Biosystems 7500 Prism Sequence Detection System. The results were normalized to GAPDH RNA levels, and fold change in samples was calculated as 2) as described. In a separate experiment, dcSSc fibroblasts were treated with BET inhibitor JQ1 for 48 h before the cells were collected for mRNA extraction. After cDNA synthesis, the samples were subjected to qPCR using ViiA 7 Real-Time PCR System.
. In other experiments, whole-cell lysates prepared from TGF-β treated confluent cultures of wild-type and A20MEFs were immunoprecipitated with antibodies to P4D1 followed by immunoblotting using antibodies to ubiquitin TRAF6 and P4D1.Subconfluent fibroblasts in culture were co-transfected with A20-luc, NF-κB mutated A20-luc, and Renilla luciferase pRL-TK plasmids using Lipofectamine reagent .
Immunofluorescence confocal cytochemistry using skin, lung fibroblasts, and mouse embryonic fibroblasts Fibroblasts seeded on 8-well Lab-Tek II chamber glass slides were incubated in serum-free DMEM with or without TGF-β for 24 h. Cells were then fixed with 4% paraformaldehyde , permeabilized with 0.
. Nuclei were identified using DAPI, and immunofluorescence was evaluated under Nikon A1C confocal microscope.The sample size was estimated based on our previous experiments for both human and mouse studies. No statistical method was used to predetermine sample size as for both human and mouse studies enough samples were available at the same time.
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