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Results: 9

1.
Figure 2

Figure 2. Relative phosphorylation of KSHV LANA and EBV EBNA1.. From: Phosphorylation of the Chromatin Binding Domain of KSHV LANA.

A. Histogram comparing the number of human kinases phosphorylating individual EBV proteins on the protein array with the number recognizing KSHV LANA and EBV EBNA1. B. Histogram comparing the number of serine (S), threonine (T) and tyrosine (Y) residues in individual EBV proteins with the number in KSHV LANA and EBV EBNA1.

Crystal Woodard, et al. PLoS Pathog. 2012 October;8(10):e1002972.
2.
Figure 8

Figure 8. LANA protein loss is post-transcriptional and leads to p21 induction.. From: Phosphorylation of the Chromatin Binding Domain of KSHV LANA.

Comparison of LANA protein (A,B) and mRNA levels (C) with p21 mRNA induction (D) in BC3 and BCBL1 PEL cells treated for 1, 2 or 3 days with 1.7 µM BRD 7389. Protein levels were quantified using Image J and RNA was quantified using real time RT-PCR.

Crystal Woodard, et al. PLoS Pathog. 2012 October;8(10):e1002972.
3.
Figure 1

Figure 1. Phosphorylation assays on the EBV plus LANA protein array.. From: Phosphorylation of the Chromatin Binding Domain of KSHV LANA.

A. Protein array probed with anti-GST antibody followed by CY5-labeled secondary antibody. Note that the EBNA1 (392–641)-V5-6xHis, 6xHis-Biotin AviTag-LANA (1–329) and control proteins are not expressed as GST-fusions and are not detected with anti-GST antibody. B. Control incubation with [γ32P]-ATP in kinase buffer. C. Incubation with [γ32P]-ATP, kinase buffer plus casein kinase 1, gamma 2 (CSNK1G2). Boxed signal, NME1 kinase control.

Crystal Woodard, et al. PLoS Pathog. 2012 October;8(10):e1002972.
4.
Figure 3

Figure 3. Phosphomimetic mutations of LANA S10 and T14 rescue histone binding.. From: Phosphorylation of the Chromatin Binding Domain of KSHV LANA.

A. Western blots showing the effect on binding to histone H2B of individual alanine substitutions across the LANA chromatin binding domain. Flag-LANA was immunoprecipitated from transfected HEK293T cells and the bound endogenous H2B was detected using anti-H2B antibody. B. Western blots showing the effect of individual and grouped phosphomimetic mutations at S10, S13 and T14 on LANA binding to H2B. Flag-LANA or Flag-LANA deleted for the central repeats (dCR) was immunoprecipitated from transfected HEK293T cells and the bound endogenous H2B was detected using anti-H2B antibody.

Crystal Woodard, et al. PLoS Pathog. 2012 October;8(10):e1002972.
5.
Figure 9

Figure 9. Treatment of PEL cells with BRD 7389 decreases cell proliferation and increases PARP cleavage.. From: Phosphorylation of the Chromatin Binding Domain of KSHV LANA.

A. Growth of BC3 PEL and BJAB B cell lines after treatment with the indicated doses of RSK inhibitor. B. Western blot comparing the levels of cleaved PARP in BJAB B cells and BC3 and BCBL1 PEL cells after treatment for 1 day with 0.85 µM RSK inhibitor. C. Western blot comparing the levels of cleaved (*) and uncleaved PARP in BJAB, BC3 and BCBL1 cells after treatment with 0.4 or 1.7 µM RSK inhibitor.

Crystal Woodard, et al. PLoS Pathog. 2012 October;8(10):e1002972.
6.
Figure 4

Figure 4. Phosphorylation of GST-LANA(1–50).. From: Phosphorylation of the Chromatin Binding Domain of KSHV LANA.

A. Diagram of the GST-LANA aa 1–50 wild-type (1) and mutant constructions (2–5) showing the amino acid changes at positions 10, 13 and 14 in the chromatin binding domain. B. Coomassie brilliant blue staining of the purified GST-LANA(1–50) wild-type (1) and mutant (2–5) proteins used in the phosphorylation assays. C. Examples of in vitro phosphorylation assays in which GST-LANA(1–50) wild-type and mutant proteins were incubated with CSNK1G2 or MAPK14/p38a kinases. (−), minus peptide.

Crystal Woodard, et al. PLoS Pathog. 2012 October;8(10):e1002972.
7.
Figure 6

Figure 6. Impairment of LANA binding to H2B occurs upon short-term treatment with RSK inhibitor but not inhibitors of CKI, PIM1 or GSK-3.. From: Phosphorylation of the Chromatin Binding Domain of KSHV LANA.

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%.

Crystal Woodard, et al. PLoS Pathog. 2012 October;8(10):e1002972.
8.
Figure 7

Figure 7. Longer exposure to RSK inhibitor decreases LANA protein levels.. From: Phosphorylation of the Chromatin Binding Domain of KSHV LANA.

A. Western blot of Flag-LANA transfected cells showing that the further reduction in LANA interaction with endogenous histone H2B that occurs upon long-term inhibitor exposure is co-incident with a decrease in Flag-LANA protein levels. B. Western blot comparing the effect of 24 hr exposure to RSK inhibition on LANA levels in wt Flag-LANA or Flag-LANA [S10E, S14E, T14E] transfected cells. C. Western blots showing the effect of increasing doses of BRD 7389 on LANA protein levels in PEL cells. D. Western blot showing the effect of the proteasome inhibitor lactacystin on LANA protein levels in BCBL1 cells treated with RSK inhibitor.

Crystal Woodard, et al. PLoS Pathog. 2012 October;8(10):e1002972.
9.
Figure 5

Figure 5. Identification of kinases that phosphorylate the LANA chromatin binding domain.. From: Phosphorylation of the Chromatin Binding Domain of KSHV LANA.

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.

Crystal Woodard, et al. PLoS Pathog. 2012 October;8(10):e1002972.

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