Figure 3
From: Negative regulation of NF-κB signaling in T lymphocytes by the ubiquitin-specific protease USP34
Silencing of USP34 enhances IκBα degradation and NF-κB binding to DNA. Jurkat cells were transfected with non-targeting- (n.t.) or USP34-targeting siRNA for 72-96 h. (A) Lysates from cells stimulated with 40 ng.ml-1 PMA plus 300 ng.ml-1 ionomycin (P/I) for 0, 10, 30 and 60 min were analyzed by immunoblot as indicated. M.W., molecular weight markers. (B) NF-κB binding to DNA was assessed on nuclear extracts from cells stimulated as in (A) for the indicated time by non-radioactive electrophoretic mobility shift assays (EMSA) using biotin-labeled κB element DNA sequence. Note that DNA:p65 complexes were chased away with a cold probe, and were shifted when a p65 antibody was added. (C) Nuclear and cytoplasmic extracts from cells stimulated as in (A) were analyzed by immunoblot as indicated. GAPDH and PARP served as loading controls. (D) Lysates from cells stimulated as in (A) were assessed by immunoblot as indicated.