sipkr  (Santa Cruz Biotechnology)

Journal: Journal of Virology

Article Title: A Map of the Arenavirus Nucleoprotein-Host Protein Interactome Reveals that Junín Virus Selectively Impairs the Antiviral Activity of Double-Stranded RNA-Activated Protein Kinase (PKR)

doi: 10.1128/JVI.00763-17

Figure Lengend Snippet: Loss of functional PKR does not affect arenavirus propagation. A549 cells were transfected with a nontargeting scrambled siRNA (siSCR) or one of two PKR-specific siRNAs (siPKR #1 and siPKR #2). Three days after siRNA transfection, cells were infected with JUNV (A to C) or LCMV (D to F), and supernatants and cellular protein lysates were collected at 48 hpi (JUNV and LCMV) and 72 hpi (JUNV only). (A and D) Protein levels of PKR and viral NP were visualized by Western blotting. (B and E) NP protein levels were quantified, normalized to β-actin levels, and normalized to the mean NP level in siSCR-transfected cells at 48 hpi and then compared by one-way ANOVA. (C and F) Quantities of infectious virus in supernatants were determined by plaque assay and were compared by one-way ANOVA. Data are presented as mean PFU per milliliter ± SEM for 2 independent experiments featuring 3 technical replicates each for panels A to C (JUNV) or for 3 independent experiments featuring 3 technical replicates each for panels D to F (LCMV). ns, not significant (P > 0.05); **, P ≤ 0.01.

Article Snippet: The following Silencer Select siRNAs were used for knockdown experiments: Silencer Select negative control 1 siRNA siSCR (Thermo Fisher), siPKR-1 (s11185; Thermo Fisher) ( and ), and siPKR-2 (s229501; Thermo Fisher) ( ).

Techniques: Functional Assay, Transfection, Infection, Western Blot, Plaque Assay

Journal: Journal of Virology

Article Title: A Map of the Arenavirus Nucleoprotein-Host Protein Interactome Reveals that Junín Virus Selectively Impairs the Antiviral Activity of Double-Stranded RNA-Activated Protein Kinase (PKR)

doi: 10.1128/JVI.00763-17

Figure Lengend Snippet: Loss of functional PKR does not affect arenavirus propagation. A549 cells were transfected with a nontargeting scrambled siRNA (siSCR) or one of two PKR-specific siRNAs (siPKR #1 and siPKR #2). Three days after siRNA transfection, cells were infected with JUNV (A to C) or LCMV (D to F), and supernatants and cellular protein lysates were collected at 48 hpi (JUNV and LCMV) and 72 hpi (JUNV only). (A and D) Protein levels of PKR and viral NP were visualized by Western blotting. (B and E) NP protein levels were quantified, normalized to β-actin levels, and normalized to the mean NP level in siSCR-transfected cells at 48 hpi and then compared by one-way ANOVA. (C and F) Quantities of infectious virus in supernatants were determined by plaque assay and were compared by one-way ANOVA. Data are presented as mean PFU per milliliter ± SEM for 2 independent experiments featuring 3 technical replicates each for panels A to C (JUNV) or for 3 independent experiments featuring 3 technical replicates each for panels D to F (LCMV). ns, not significant (P > 0.05); **, P ≤ 0.01.

Article Snippet: The following Silencer Select siRNAs were used for knockdown experiments: Silencer Select negative control 1 siRNA siSCR (Thermo Fisher), siPKR-1 (s11185; Thermo Fisher) ( and ), and siPKR-2 (s229501; Thermo Fisher) ( ).

Techniques: Functional Assay, Transfection, Infection, Western Blot, Plaque Assay

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