Journal: PLoS ONE
Article Title: CK2 Phosphorylation of Schistosoma mansoni HMGB1 Protein Regulates Its Cellular Traffic and Secretion but Not Its DNA Transactions
Figure Lengend Snippet: DNA supercoiling and bending assays by phosphorylated SmHMGB1. (A) Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I with 1 µg of recombinant SmHMGB1-FL or SmHMGB1-S172A/S174A that were phosphorylated (lanes 3–5) or not (lanes 6–8 and 9–11), by CK2. Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gels with ethidium bromide. Form I, supercoiled DNA; form II, relaxed circular DNA. (B) Top panel: autoradiography; bottom panel: Coomassie staining. (C) A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with 50 ng of recombinant proteins, that were phosphorylated (lanes 7–9) or not (lanes 4–6, 10–12, 13–15 and 16–18), followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Controls are as follows: FL(c1): SmHMGB1-FL without CK2; FL(c2): SmHMGB1-FL without phosphate; FL(c3): SmHMGB1-FL without CK2 buffer. Linear: linear DNA; Lm: linear multimers. (D) Top panel: autoradiography; bottom panel: Coomassie staining. These experiments were repeated four times.
Article Snippet: Briefly, a 32 P-labeled-66-bp or a 32 P-labeled-123-bp DNA fragments (1 nM) with cohesive BamHI ends were pre-incubated on ice for 20 min with appropriate amounts of recombinant proteins (50 ng), total (10 µg), nuclear (4 µg) or cytoplasmic (4 µg) adult worm extracts, in 1× T4 DNA ligase buffer (30 mM Tris–HCl, pH 7.8, 10 mM MgCl2 , 10 mM dithiothreitol, and 0.5 mM ATP; Promega) in a final volume of 20 µl.
Techniques: Plasmid Preparation, Incubation, Recombinant, Staining, Autoradiography, Labeling, Ligation, DNA Ligation, Electrophoresis