ATM augments nuclear stabilization of DYRK2 by inhibiting MDM2 in the apoptotic response to DNA damage - PubMed (original) (raw)

DYRK2 degradation is associated with MDM2-mediated polyubiquitination. A, 293T cells expressing FLAG-DYRK2 were treated with ADR for 24 h in the presence of 1 μ

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MG-132. Cell lysates were immunoprecipitated (IP) with anti-FLAG-agarose. The immunoprecipitates were analyzed by immunoblotting (IB) with anti-ubiquitin (top panel), anti-MDM2 (second panel), or anti-FLAG (third panel). Lysates were also analyzed by immunoblotting with anti-lamin B1 (bottom panel). B, in vitro ubiquitination assays were performed and analyzed by immunoblotting with anti-ubiquitin (upper panel) or anti-His (lower panel). C, 293T cells ectopically expressing FLAG-DYRK2 were transfected with scramble siRNA or MDM2 siRNA followed by treatment with MG-132. Nuclear lysates were immunoprecipitated with anti-FLAG-agarose. The immunoprecipitates were analyzed by immunoblotting with anti-ubiquitin (top panel) or anti-FLAG (second panel). Lysates were also analyzed by immunoblotting with anti-MDM2 (third panel), anti-FLAG (fourth panel), or anti-lamin B1 (bottom panel). D, 293T cells were transfected with GFP-DYRK2 and/or MDM2 siRNA. The cells were left untreated or treated with 2 μ

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MG-132 for 4 h. Cells were stained with anti-GFP or anti-MDM2. The nuclei were stained with DAPI. E, 293T cells were transfected with scramble siRNA or MDM2 siRNA followed by treatment with MG-132. Cell lysates were subjected to immunoblot analysis with anti-DYRK2 (upper panel), anti-MDM2 (middle panel), or anti-tubulin (lower panel). A ratio of DYRK2 expression was determined by the densitometric analysis using the ImageJ program. The expression in MG-132-treated cells transfected with scramble siRNA was defined as 1.00. F, 293T cells ectopically expressed with FLAG-DYRK2 were transfected with scramble siRNA or MDM2 siRNA. Lysates were analyzed by immunoblotting with the indicated antibodies. G, 293T cells were transfected with scramble siRNA or MDM2 siRNA and then treated with 20 μ

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etoposide for 24 h. Lysates were analyzed by immunoblotting with the indicated antibodies. H, 293T cells were treated with MG-132. Cell lysates were immunoprecipitated with normal IgG or anti-MDM2 followed by immunoblot analysis with anti-DYRK2 (upper panel) or anti-MDM2 (lower panel). I, 293T cells expressing FLAG-DYRK2 were treated with 20 μ

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etoposide for 24 h. Cell lysates were immunoprecipitated with anti-FLAG-agarose. The immunoprecipitates were analyzed by immunoblotting with anti-MDM2 (top panel) or anti-FLAG (second panel). Lysates were also analyzed by immunoblotting with anti-FLAG (third panel), anti-MDM2 (fourth panel), or anti-tubulin (bottom panel).