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: PLoS ONE

Article Title: Involvement of Activation of PKR in HBx-siRNA-Mediated Innate Immune Effects on HBV Inhibition

doi: 10.1371/journal.pone.0027931

Figure Lengend Snippet: (A) HepG2.2.15 cells were treated with or without PKR inhibitor C16, and then transfection experiments were performed for 24 h. Levels of IFN-α and IFN-β mRNA were analyzed by real-time PCR and presented relative to mock transfection (left). The levels of IFN-α in supernatants were examined by ELISA (right). (B) The experiment was performed as . Levels of IFN-stimulated gene (ISG)15 and ISG56 mRNA were analyzed by real-time PCR and presented relative to mock transfection. (C) p-Stat1 expression was detected by flow cytometry when siRNA was transfected for 4 h. (D) The experiment was performed as , mRNA levels of TNF-α and IL-6 were analyzed by quantitative real-time PCR and were presented relative to mock transfection. (E) siPKR were transfected into cells, then PKR protein expression was assayed by Western Blot (top). siRNA4 and siRNA targeting PKR were cotransfected into HepG2.2.15 cells for 24 h, then mRNA levels of IFN-α and IFN-β were analyzed and presented relative to mock transfection (bottom). Data are expressed as the mean ± SD from at least three separate experiments. * p <0.05 versus siRNA4-treated group.

Article Snippet: The target sequences of PKR-siRNA (siPKR) were: GAA CUG CCU AAU UCA GGA C,and were synthesized by Ribo Company (RiboBio, Guangzhou, China).

Techniques: Transfection, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Western Blot

Journal: PLoS ONE

Article Title: Involvement of Activation of PKR in HBx-siRNA-Mediated Innate Immune Effects on HBV Inhibition

doi: 10.1371/journal.pone.0027931

Figure Lengend Snippet: HepG2.2.15 cells were treated with or without C16, and then transfection experiments were performed at a concentration of 150 nM. Levels of HBx RNA mRNA ( A) and HBsAg proteins in supernatants (C) were examined as described previously. Levels were expressed as percentages of corresponding levels in scramble siRNA-treated cells. HBV DNA in supernatants was detected by real-time PCR (B). Immunostaining for HBcAg were visualized under a microscope (D). siRNA4 and siPKR were cotransfected into HepG2.2.15 cells, then levels of HBx mRNA were analyzed by quantitative real-time PCR and presented relative to mock transfection (E). Data are expressed as the mean ± SD from at least three independent experiments. * p <0.05 versus corresponding solvent-treated group.

Article Snippet: The target sequences of PKR-siRNA (siPKR) were: GAA CUG CCU AAU UCA GGA C,and were synthesized by Ribo Company (RiboBio, Guangzhou, China).

Techniques: Transfection, Concentration Assay, Real-time Polymerase Chain Reaction, Immunostaining, Microscopy, Solvent

Journal: Molecular Vision

Article Title: PKR promotes choroidal neovascularization via upregulating the PI3K/Akt signaling pathway in VEGF expression

doi:

Figure Lengend Snippet: Effect of PKR siRNA and PI3K inhibitor LY294002 on hypoxia-induced PKR, p-PI3K, p-Akt, and VEGF expression in RF/6A cells. A : DsRNA-activated protein kinase (PKR), phosphophosphatidylinositol 3-kinase (p-PI3K), phosphoprotein kinase B (p-Akt), and vascular endothelial growth factor (VEGF) expression was detected with western blotting in the RF/6A cells following PKR siRNA transfection after 48 h. C : The histogram shows the densitometric analysis of the average levels for PKR, p-PI3K, p-Akt, and VEGF to GAPDH. Cells transfected with scramble siRNA were used as the negative control. B : The western blot shows PKR, p-PI3K, p-Akt, and VEGF expression in RF/6A cells treated with the PI3K inhibitor LY294002 for 30 min. D : The histogram shows the densitometric analysis of the average levels of PKR, p-PI3K, p-Akt, and VEGF to GAPDH. Hypoxic cells were used as the negative control. *p<0.05, statistically significantly different compared to the respective controls. Values represent means ± SD.

Article Snippet: Transfection of PKR siRNA (5′-GGA TTC GGG TTA CTT GTA A-3′, RiboBio, Guangzhou, China) was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol.

Techniques: Expressing, Western Blot, Transfection, Negative Control

Journal: Molecular Vision

Article Title: PKR promotes choroidal neovascularization via upregulating the PI3K/Akt signaling pathway in VEGF expression

doi:

Figure Lengend Snippet: PKR promotes migration of RF/6A cells in a coculture system under hypoxic conditions. A : Crystal violet staining detected the migrated cells of each group: normal, hypoxic, scramble siRNA, and dsRNA-activated protein kinase (PKR) siRNA. Representative photographs of migrated RF/6A cells (200× magnification). B : The average number of migrated RF/6A cells per field. *p<0.01, PKR siRNA group versus the hypoxic group. Values represent means ± SD.

Article Snippet: Transfection of PKR siRNA (5′-GGA TTC GGG TTA CTT GTA A-3′, RiboBio, Guangzhou, China) was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol.

Techniques: Migration, Staining

Journal: Molecular Vision

Article Title: PKR promotes choroidal neovascularization via upregulating the PI3K/Akt signaling pathway in VEGF expression

doi:

Figure Lengend Snippet: PKR promotes tube formation of RF/6A cells in a coculture system under hypoxic conditions. A : Microscopic images showing tube formation in each group: normal, hypoxic, scramble siRNA, and dsRNA-activated protein kinase (PKR) siRNA. Representative photographs of tube formation of RF/6A cells (200× magnification). B : Decreased tube formation was observed when the cocultured RF/6A cells were transfected with PKR siRNA under hypoxia. *p<0.01, PKR siRNA group versus the hypoxia group. Values represent means ± SD.

Article Snippet: Transfection of PKR siRNA (5′-GGA TTC GGG TTA CTT GTA A-3′, RiboBio, Guangzhou, China) was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol.

Techniques: Transfection