pflag cmv constructs  (Addgene inc)

Journal: Cell Cycle

Article Title: DNA damage-induced regulatory interplay between DAXX, p53, ATM kinase and Wip1 phosphatase

doi: 10.4161/15384101.2014.988019

Figure Lengend Snippet: DAXX depletion or S564A mutation does not affect Mdm2/p53 stability or p53‑mediated gene expression. ( A ) BJ fibroblasts stably transduced with empty lentiviral pCDH vector or either pCDH-DAXX WT or pCDH-DAXX S564A were treated with 40 μM VP16 for 0, 2, 4 or 6 hours and RNA expression of the indicated p53-dependent genes was analyzed by quantitative RT‑PCR. Expression values were normalized to the average of 3 reference genes (β-actin, SDH and ALAS). ( B ) Transduced BJ fibroblast as in ( A ) were exposed to 40 μM VP16 for the indicated times and subjected to protein gel blotting analysis using add p53 - antibodies against DAXX, phospho-p53 (S15), p53 or p21. ( C ) U2OS cells transfected with pXJ41 Hdm2 (human Mdm2) together with empty FLAG-CMV, FLAG-DAXX WT or FLAG-DAXX S564A were treated with 50 μl/ml CHX alone or together with 10 μM VP16 for the specified time points. Cell were harvested and lysates separated by SDS–PAGE and probed with indicated antibodies. ( D ) BJ fibroblasts were depleted by control siRNA (siLuc) or siRNA against Wip1 and 3 d after transfection treated with 4 nM NCS. Cells were lysed at the indicated time points after DNA damage and analyzed by western blotting using labeled antibodies. GAPDH was used as a loading control.

Article Snippet: pFLAG-CMV-DAXX WT construct was generated by PCR and cloning into pFLAG-CMV-5a vector (Sigma Aldrich) and confirmed by sequencing.

Techniques: Mutagenesis, Expressing, Stable Transfection, Transduction, Plasmid Preparation, RNA Expression, Quantitative RT-PCR, Transfection, SDS Page, Western Blot, Labeling