Journal: Brain Pathology

Article Title: Modulation of Tau Phosphorylation by the Kinase PKR: Implications in Alzheimer's Disease

doi: 10.1111/j.1750-3639.2010.00437.x

Figure Lengend Snippet: Effect of PKR inhibitors (PRI and C16) on tunicamycin (Tm) induced activation of PKR and GSK‐3β in SH‐SY5Y cells. A. Untreated neuroblastoma cell lines showing slight staining of pPKRThr446 (green) and pGSK‐3βTyr216 (red) in the cytoplasm, without apoptotic nuclei. After 8 h of Tm (5 µg/mL) treatment, the labeling of pPKRThr451 and pGSK‐3βTyr216 and their co‐localization were increased in the cytoplasm and nuclei. Adding PRI (50 µM) or C16 (1 µM) after 8 h of Tm exposure lead to an attenuation of the activation of PKR and GSK‐3β in the cytoplasm and nucleus associated with a strong reduction of co‐localization. Horizontal bar 10 µM. B. The cell counting confirmed that PKR (50%) and GSK‐3β (20%) activated after 8 h of Tm treatment and it increases, respectively, to 75% and 80% after 16 h. C16 attenuates both nuclear activation of PKR and GSK‐3β.

Article Snippet: The PRI (peptide PKR inhibitor) (NeoMPS Polypeptide Laboratories, Strasbourg, France) can bind to the double‐stranded RNA‐binding site of PKR and prevent its activation (25) .

Techniques: Activation Assay, Staining, Labeling, Cell Counting

Journal: Brain Pathology

Article Title: Modulation of Tau Phosphorylation by the Kinase PKR: Implications in Alzheimer's Disease

doi: 10.1111/j.1750-3639.2010.00437.x

Figure Lengend Snippet: Immunoblot analysis of Tm treatment in SH‐SY5Y cells with or without PRI peptide. A. A progressive activation of PKR which peaks after 4 h and GSK‐3β, with highest levels after 8 h. Both are reduced at the different time of treatment by the PRI inhibitor. PARP cleavage progressively increased over time after Tm exposure and PRI peptide exposure reduced PARP cleavage at 2, 4 and 8 h. Results were obtained from 5 independent experiments. *P < 0.05, **P < 0.01. B. Tm has opposite effect on GSK‐3β phosphorylation on tyrosine 216 and serine 9, with a reduction of the level of pGSK3βser9, partially reverses by PRI addition. Results were obtained from 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. C. Analysis of prepared cytoplasmic and nuclear fractions from SH‐SY5Y cells revealed that GSK‐3β begins to be translocated into the nucleus from 4 h of Tm treatment. This translocation is maximum after 8 h and with consequent increase in the phosphorylation at 4 and 8 h. Nuclear activation is attenuated by the PRI at 4 and 8 h. The evaluation of cell fractionation was assessed for cytosolic fraction with anti‐KDEL and, for nuclear fraction with anti‐histone H3.

Article Snippet: The PRI (peptide PKR inhibitor) (NeoMPS Polypeptide Laboratories, Strasbourg, France) can bind to the double‐stranded RNA‐binding site of PKR and prevent its activation (25) .

Techniques: Western Blot, Activation Assay, Phospho-proteomics, Translocation Assay, Cell Fractionation

Journal: Brain Pathology

Article Title: Modulation of Tau Phosphorylation by the Kinase PKR: Implications in Alzheimer's Disease

doi: 10.1111/j.1750-3639.2010.00437.x

Figure Lengend Snippet: Tm induces phosphorylation of tau in SH‐SY5Y cells, attenuated by PRI addition. A. Immunoblot analysis with anti‐Tau, anti‐pTau (AT8) antibodies showed that Tm induces the phosphorylation of Tau at AT8 epitope with highest levels after 4 h of treatment and this phosphorylation is reduced by the PRI inhibitor. B. Immunoblot analysis of Tau phosphorylation on three other epitopes shows that Tm leads to phosphorylation at 2 h and 4 h of AT100 and AT270 but not AT180 tau. Results were obtained from 5 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The PRI (peptide PKR inhibitor) (NeoMPS Polypeptide Laboratories, Strasbourg, France) can bind to the double‐stranded RNA‐binding site of PKR and prevent its activation (25) .

Techniques: Phospho-proteomics, Western Blot

Journal: Brain Pathology

Article Title: Modulation of Tau Phosphorylation by the Kinase PKR: Implications in Alzheimer's Disease

doi: 10.1111/j.1750-3639.2010.00437.x

Figure Lengend Snippet: Immunoblot analysis of Aβ1‐42 effect on GSK‐3β, PKR and Tau phosphorylation. SH‐SY5Y cells were pretreated with or without 50 µM PRI peptide, and then exposed to Aβ for 4 h or 8 h. The blots showing that Aβ induces the phosphorylation of PKR, GSK‐3β and Tau which gradually increases after 4 and 8 h of treatment and this activation is significantly reduced (35 to 40%) by the PRI inhibitor. Results were obtained from 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The PRI (peptide PKR inhibitor) (NeoMPS Polypeptide Laboratories, Strasbourg, France) can bind to the double‐stranded RNA‐binding site of PKR and prevent its activation (25) .

Techniques: Western Blot, Phospho-proteomics, Activation Assay

Journal: Frontiers in Microbiology

Article Title: Duck Hepatitis A Virus Type 1 Induces eIF2α Phosphorylation-Dependent Cellular Translation Shutoff via PERK/GCN2

doi: 10.3389/fmicb.2021.624540

Figure Lengend Snippet: PERK and GCN2 are involved in eIF2α phosphorylation during DHAV-1 infection. (A) Screen the kinases that affect eIF2α phosphorylation. DEFs were infected with DHAV-1 at MOI of 1. After 22 h of infection, different concentrations of kinase inhibitors were added to DEFs for 2 h. Then, DEFs were harvested for immunoblot analysis with the indicated antibodies. (B) PERK inhibitor GSK2606414 inhibits eIF2α phosphorylation induced by DHAV-1. (C) GCN2 inhibitor GCN2-IN-1 inhibits eIF2α phosphorylation induced by DHAV-1. (D) PKR inhibitor C16 cannot inhibit eIF2α phosphorylation induced by DHAV-1. (E) Transfection of poly(I:C) activate PKR kinase. (F) DHAV-1 and poly(I:C) stimulate PKR transcription. Differences between two groups were analyzed using Student’s t -test and considered as significant at * p < 0.05, ** p < 0.01, and *** p < 0.001. The bands marked by asterisk (*) are non-specific proteins.

Article Snippet: C16 (PKR Inhibitor) and GSK2606414 (PERK inhibitor) were purchased from APExBIO, and GCN2-IN-1 (GCN2 inhibitor) was purchased from MCE.

Techniques: Phospho-proteomics, Infection, Western Blot, Transfection

Journal: Brain Pathology

Article Title: The PKR Activator PACT Is Induced by Aβ: Involvement in Alzheimer's Disease

doi: 10.1111/j.1750-3639.2011.00520.x

Figure Lengend Snippet: A, B and C immunoblot analysis of Aβ exposure and PRI peptide in SH‐SY5Y cells. PACT and pPKRthr446 progressively increased over time after Aβ1‐42 treatment with peaks after 8 h. PKR activation is decreased with PRI treatment, but not PACT expression, while full PKR and tubulin are stable. Results were obtained from five independent experiments (*P < 0.05; **P < 0.001; ***P < 0.0001). D, E and F PACT and pPKRthr446 levels are stable after nontoxic Aβ42‐1 treatment that supports specific role of Aβ1‐42 in enhanced PACT/PKR interaction.

Article Snippet: Reagents Inhibitor of PKR: The PRI (NeoMPS Polypeptide Laboratories, Strasbourg, France) can bind to the double‐stranded RNA binding site of PKR and prevent its activation (20) .

Techniques: Western Blot, Activation Assay, Expressing

Journal: Brain Pathology

Article Title: The PKR Activator PACT Is Induced by Aβ: Involvement in Alzheimer's Disease

doi: 10.1111/j.1750-3639.2011.00520.x

Figure Lengend Snippet: A, B and C immunoblot analysis of Aβ exposure and PRI peptide in SH‐SY5Y cells. PACT and pPKRthr446 progressively increased over time after Aβ1‐42 treatment with peaks after 8 h. PKR activation is decreased with PRI treatment, but not PACT expression, while full PKR and tubulin are stable. Results were obtained from five independent experiments (*P < 0.05; **P < 0.001; ***P < 0.0001). D, E and F PACT and pPKRthr446 levels are stable after nontoxic Aβ42‐1 treatment that supports specific role of Aβ1‐42 in enhanced PACT/PKR interaction.

Article Snippet: Reagents Inhibitor of PKR: The PRI (NeoMPS Polypeptide Laboratories, Strasbourg, France) can bind to the double‐stranded RNA binding site of PKR and prevent its activation (20) .

Techniques: Western Blot, Activation Assay, Expressing