Journal:

Article Title: PKR Regulates B56?-mediated BCL2 Phosphatase Activity in Acute Lymphoblastic Leukemia-derived REH Cells *

doi: 10.1074/jbc.M800951200

Figure Lengend Snippet: Suppression of PKR expression by shRNA suppresses B56α expression and promotes BCL2 phosphorylation. A, Western blot analysis was performed using antibodies against PKR, B56α, p-eIF2 α, eIF2 α, BCL2, and actin on total lysate (1 × 106 cell equivalents) from REH cells transfected with control shRNA or PKR shRNA. B, REH cells transfected with control shRNA or REH cells transfected with PKR shRNA were labeled with [32P]orthophosphoric acid (32P). BCL2 was immunoprecipitated using polyclonal rabbit antisera (Santa Cruz Biotechnology), electrophoresed using 10% SDS-PAGE, and transferred to a nitrocellulose filter, and phosphorylated bands were detected by autoradiography. The identity of the BCL2 32P-labeled band was established by using a mouse monoclonal antibody against BCL2 (Dako) on the same filter. C, Western blot analysis was performed using antibodies against B55, PP2A/A, and GAPDH on total lysate (1 × 106 cell equivalents) from REH cells transfected with control shRNA or PKR shRNA.

Article Snippet: A shRNA plasmid targeting PKR (designated TI379192) is commercially available from OriGene.

Techniques: Expressing, shRNA, Phospho-proteomics, Western Blot, Transfection, Control, Labeling, Immunoprecipitation, SDS Page, Autoradiography

Journal:

Article Title: PKR Regulates B56?-mediated BCL2 Phosphatase Activity in Acute Lymphoblastic Leukemia-derived REH Cells *

doi: 10.1074/jbc.M800951200

Figure Lengend Snippet: Suppression of PKR expression by shRNA suppresses mitochondrial PP2A activity and promotes chemoresistance. A, mitochondrial (Mito) and nuclear (Nuc) PP2A activity was determined using a molybdate dye assay for REH cells transduced with control shRNA or with shRNA against PKR. Error bars represent the mean ± S.D. from three separate experiments. *, statistically significant differences from PP2A activity from REH cells transduced with control shRNA (p < 0.05, standard t test). B, REH parental cells were treated with 10 μm PKR inhibitor for 3 h, and mitochondrial PP2A activity was determined using the molybdate dye assay. Error bars represent the mean ± S.D. from three separate experiments. *, statistically significant differences from PP2A activity in untreated REH cells (p < 0.05, standard t test). C, Western blot analysis was performed to determine PKR activity in untreated REH cells and cells treated with 10 μm Calbiochem PKR inhibitor for 3 or 20 h using antibody against p-PKR. PKR antibody was used to measure total kinase.

Article Snippet: A shRNA plasmid targeting PKR (designated TI379192) is commercially available from OriGene.

Techniques: Expressing, shRNA, Activity Assay, Transduction, Control, Western Blot

Journal:

Article Title: PKR Regulates B56?-mediated BCL2 Phosphatase Activity in Acute Lymphoblastic Leukemia-derived REH Cells *

doi: 10.1074/jbc.M800951200

Figure Lengend Snippet: Suppression of PKR expression by shRNA promotes chemoresistance. Apoptosis of shRNA-transduced REH cells treated with 1 μm etoposide for 24 h was examined using FACScan analysis of sub-G0 content of propidium iodide-stained cells. Error bars represent the mean ± S.D. from three separate experiments. *, statistically significant differences from cell viability in untreated REH cells (p < 0.05, standard t test).

Article Snippet: A shRNA plasmid targeting PKR (designated TI379192) is commercially available from OriGene.

Techniques: Expressing, shRNA, Staining

Journal:

Article Title: PKR Regulates B56?-mediated BCL2 Phosphatase Activity in Acute Lymphoblastic Leukemia-derived REH Cells *

doi: 10.1074/jbc.M800951200

Figure Lengend Snippet: eIF2α and AKT promote B56α protein expression. A, Western blot analysis was performed to determine the effect of salubrinal on B56α expression. HA antibody was used to detect HA-GFP protein in REH/GFP cells and exogenous HA-B56α protein in REH/WT B56α cells on total lysates (1 × 106 cell equivalents) from untreated cells or cells treated with 75 μm salubrinal for 24 h. Antibodies against eIF2α, p-eIF2α, and tubulin were used as controls. B, Western blot analysis was performed to determine the effect of LY294002 on B56α expression in REH cells transfected with control shRNA and REH cells transfected with PKR shRNA. Rabbit polyclonal antibody against B56α protein was used on total lysates (1 × 106 cell equivalents) from untreated cells or cells treated with 1 μm LY294002 for 24 h. Antibodies against p-AKT, AKT, and tubulin were used as controls.

Article Snippet: A shRNA plasmid targeting PKR (designated TI379192) is commercially available from OriGene.

Techniques: Expressing, Western Blot, Transfection, Control, shRNA

Journal: Journal of Virology

Article Title: Conserved Cx n C Motifs in Kaposi’s Sarcoma-Associated Herpesvirus ORF66 Are Required for Viral Late Gene Expression and Are Essential for Its Interaction with ORF34

doi: 10.1128/jvi.01299-19

Figure Lengend Snippet: Figure 1. ORF66 is essential in KSHV and required for late gene transcription 600

Article Snippet: ORF66 aa 1-200 was cloned into the NotI and XhoI sites 357 of pcDNA4/TO-2xStrep (N-terminal tag) to generate pcDNA4/TO-2xStrep-ORF66 1-200 358 (Addgene plasmid #130954) and ORF66 aa 200-429 was cloned into the BamHI and XhoI sites 359 of pcDNA4/TO-2xStrep (C-terminal tag) to generate pcDNA4/TO-ORF66 200-429-2xStrep 360 (Addgene plasmid #130955).

Techniques:

Journal: Journal of Virology

Article Title: Conserved Cx n C Motifs in Kaposi’s Sarcoma-Associated Herpesvirus ORF66 Are Required for Viral Late Gene Expression and Are Essential for Its Interaction with ORF34

doi: 10.1128/jvi.01299-19

Figure Lengend Snippet: Figure 3. The C-terminal domain of ORF66 is essential for protein-protein interactions 627

Article Snippet: ORF66 aa 1-200 was cloned into the NotI and XhoI sites 357 of pcDNA4/TO-2xStrep (N-terminal tag) to generate pcDNA4/TO-2xStrep-ORF66 1-200 358 (Addgene plasmid #130954) and ORF66 aa 200-429 was cloned into the BamHI and XhoI sites 359 of pcDNA4/TO-2xStrep (C-terminal tag) to generate pcDNA4/TO-ORF66 200-429-2xStrep 360 (Addgene plasmid #130955).

Techniques: Protein-Protein interactions

Journal: Journal of Virology

Article Title: Conserved Cx n C Motifs in Kaposi’s Sarcoma-Associated Herpesvirus ORF66 Are Required for Viral Late Gene Expression and Are Essential for Its Interaction with ORF34

doi: 10.1128/jvi.01299-19

Figure Lengend Snippet: Figure 5. The CxnC motifs in ORF66 are required for interaction with ORF34 652

Article Snippet: ORF66 aa 1-200 was cloned into the NotI and XhoI sites 357 of pcDNA4/TO-2xStrep (N-terminal tag) to generate pcDNA4/TO-2xStrep-ORF66 1-200 358 (Addgene plasmid #130954) and ORF66 aa 200-429 was cloned into the BamHI and XhoI sites 359 of pcDNA4/TO-2xStrep (C-terminal tag) to generate pcDNA4/TO-ORF66 200-429-2xStrep 360 (Addgene plasmid #130955).

Techniques:

Journal: Communications Biology

Article Title: Characterization of exoribonuclease XRN1 as a cancer target and identification of adenosine-3’,5’-bisphosphate as a potent enzyme inhibitor

doi: 10.1038/s42003-025-08005-y

Figure Lengend Snippet: a XRN1, pPKR, total PKR and loading control western blot for NCI-H1650, NCI-H1703, NCI-H838, and NCI-H1944 cell lines transduced with non-targeting (NT), XRN1, or POL2RL sgRNA for 7 days. b RT-qPCR for Type I interferon genes, IFNA1 and IFNB1 , 7 days post CRISPR transduction (black bars: Non-targeting control; gray bars: XRN1 knockout). Error bars represent standard deviation of three replicates. c Proliferation of NCI-H838 cells (black bars: Non-targeting control; gray bars: XRN1 knockout) following 96 hours of stimulation with exogenous IFNβ, as assessed by a luminescent assay. Error bars represent standard deviation of three replicates. d Model for XRN1 in cancers with elevated dsRNA burden and rationale for monotherapy and combination with checkpoint inhibitors.

Article Snippet: Antibodies used were as follows: XRN1 (Cell Signaling Technology (CST) 70205; 1:1000), β-Actin (CST 3700; 1:2000), total PKR (R&D systems MAB1980; 1:500), phospho-Thr451 PKR (Abcam ab81303; 1:500), IRDye 800CW Goat anti-Rabbit (Li-Cor 926-32211; 1:20,000), and IRDye 680RD Goat anti-Mouse (Licor 926-68070; 1:20,000).

Techniques: Control, Western Blot, Transduction, Quantitative RT-PCR, CRISPR, Knock-Out, Standard Deviation, Luminescence Assay