Journal: PLoS Pathogens

Article Title: Phosphorylation of the Chromatin Binding Domain of KSHV LANA

doi: 10.1371/journal.ppat.1002972

Figure Lengend Snippet: Nuclear kinases that phosphorylate N-terminal regions of LANA.

Article Snippet: For characterization of GSK-3β phosphorylation, peptide substrates were incubated for 30 min at 30°C in (i) kinase buffer with 10 µCi of [γ 32 P]-ATP, (ii) kinase buffer with 10 µCi of [γ 32 P]-ATP and Pim1 (Upstate, #14-573), (iii) kinase buffer with 10 µCi of [γ 32 P]-ATP and GSK-3β (Millipore) and for primed phosphorylations (iv) kinase buffer with 20 µM cold ATP and Pim1 (Upstate, #14-573) followed by incubation with Pim1 inhibitor SMI-4a (0.1 µM; Enzo Life Sciences) for 30 min at 30°C (Millipore).

Techniques:

Journal: PLoS Pathogens

Article Title: Phosphorylation of the Chromatin Binding Domain of KSHV LANA

doi: 10.1371/journal.ppat.1002972

Figure Lengend Snippet: A. Phosphorylation of a LANA aa3–21 peptide by the indicated kinases. The peptide was separated from the kinase reaction mixture by electrophoresis and the gel dried and subjected to autoradiography. B. Upper: Amino acid sequence of the wt LANA 3–21 peptide (1) showing the positions of the single and grouped sequence changes in the mutant peptides (2–6). Lower: Examples of phosphorylation of the wild-type and mutant peptides by the indicated kinases. C. GSK-3 phosphorylates S10 after PIM1 priming. Wild-type and S10A mutant peptides were incubated with PIM1 or GSK-3β or with PIM1 plus unlabelled γ-ATP (PIM1 priming) or with GSK-3 β after PIM1 priming followed by inhibition of PIM1 with the PIM1 inhibitor SMI-4a. (−), minus kinase.

Article Snippet: For characterization of GSK-3β phosphorylation, peptide substrates were incubated for 30 min at 30°C in (i) kinase buffer with 10 µCi of [γ 32 P]-ATP, (ii) kinase buffer with 10 µCi of [γ 32 P]-ATP and Pim1 (Upstate, #14-573), (iii) kinase buffer with 10 µCi of [γ 32 P]-ATP and GSK-3β (Millipore) and for primed phosphorylations (iv) kinase buffer with 20 µM cold ATP and Pim1 (Upstate, #14-573) followed by incubation with Pim1 inhibitor SMI-4a (0.1 µM; Enzo Life Sciences) for 30 min at 30°C (Millipore).

Techniques: Electrophoresis, Autoradiography, Sequencing, Mutagenesis, Incubation, Inhibition

Journal: PLoS Pathogens

Article Title: Phosphorylation of the Chromatin Binding Domain of KSHV LANA

doi: 10.1371/journal.ppat.1002972

Figure Lengend Snippet: A. Cells treated with CKI inhibitor (CKI-7). B. Cells treated with GSK-3 inhibitor (LiCl) or PIM1 inhibitor (SMI-4a) individually or in combination. C. Cells treated with RSK inhibitor (BRD 7389). Western blots examine interaction between transfected Flag-LANA and endogenous histone H2B. Quantitation of the band densities provides a measure of relative binding (LANA bound H2B/input H2B) and was obtained using Image J. The ratio in untreated cells was set at 100%.

Article Snippet: For characterization of GSK-3β phosphorylation, peptide substrates were incubated for 30 min at 30°C in (i) kinase buffer with 10 µCi of [γ 32 P]-ATP, (ii) kinase buffer with 10 µCi of [γ 32 P]-ATP and Pim1 (Upstate, #14-573), (iii) kinase buffer with 10 µCi of [γ 32 P]-ATP and GSK-3β (Millipore) and for primed phosphorylations (iv) kinase buffer with 20 µM cold ATP and Pim1 (Upstate, #14-573) followed by incubation with Pim1 inhibitor SMI-4a (0.1 µM; Enzo Life Sciences) for 30 min at 30°C (Millipore).

Techniques: Western Blot, Transfection, Quantitation Assay, Binding Assay