Journal: Journal of Clinical Investigation

Article Title: An anti-diabetes agent protects the mouse brain from defective insulin signaling caused by Alzheimer’s disease–associated Aβ oligomers

doi: 10.1172/jci57256

Figure Lengend Snippet: Figure 4 JNK mediates AβO-induced IRS-1pSer. (A) Representative image showing low IRS-1pSer levels (red) in a hippocampal neuron transfected with GFP-fused DN JNK (green; scale bar: 50 μm). (B) Higher-magnification image of dendrite segment (white box in A; scale bar: 10 μm). (C and D) integrated IRS-1pSer636 and IRS-1pTyr465 immunofluorescence levels, respectively. *P < 0.001 relative to vehicle-treated cultures, #P < 0.001 relative to AβO-exposed cultures; ANOVA followed by Bonferroni post-hoc test. (E–H) Hippocampal neurons exposed for 3 hours to vehicle (E), 500 nM AβOs (F), 10 μM SP600125 (SP) plus 500 nM AβOs (G), or 1 μg/ml infliximab (Inflix) plus 500 nM AβOs (H). Scale bar: 50 μm. (I) Integrated IRS-1pSer immunofluorescence levels determined from 4 experiments (independent cultures, 20 images analyzed/experi- mental condition/experiment). *P < 0.001 relative to vehicle-treated cultures, #P < 0.001 relative to AβO-exposed cultures; ANOVA followed by Bonferroni post-hoc test. Scr, scrambled Aβ1–42 peptide. (J) Immunoblot of p-JNK in cultures exposed to 500 nM AβOs for 3 hours. (K) p-JNK levels in hippocampal homogenates from APP/PS1 Tg or WT mice. tJNK, total JNK. (L) TNF-α immunoblot in concentrated conditioned medium from cultures exposed to AβOs for 3 hours. (M and N) TNF-α receptor levels in cultures exposed to 500 nM AβOs for 3 hours and in hippocampal homogenates from APP/PS1 Tg (n = 7) or WT mice (n = 5), respectively. (M and N) Densitometric quantification normalized by cyclophilin B. Lanes in J and L–N were run on the same gel but were noncontiguous. *P < 0.02, Student’s t test.

Article Snippet: Antibodies against total IRS-1 and IRS-2, IRS-1pTyr465, IRS-1pSer636, glucagon-like peptide–1 receptor (GLP1R), TNFR1, TNF-α, and the PKR inhibitor were from Santa Cruz Biotechnology Inc. IRS-1pSer307, IRS-1pSer312, and IRS-1pSer616 antibodies were from Invitrogen.

Techniques: Transfection, Immunofluorescence, Western Blot

Journal: Journal of Clinical Investigation

Article Title: An anti-diabetes agent protects the mouse brain from defective insulin signaling caused by Alzheimer’s disease–associated Aβ oligomers

doi: 10.1172/jci57256

Figure Lengend Snippet: Figure 9 Proposed mechanism underlying disrupted brain insulin signaling in AD. (A) AβOs stimulate TNF-α signaling, which activates the JNK pathway and, possibly, PKR and IKK pathways. Activation of these stress-sensitive kinases, which can also be triggered by endoplasmic reticulum stress (36), results in serine phosphorylation of IRS-1, block- ing downstream insulin signaling. (B) Stimulation of insulin and GLP1 receptors blocks AβO-induced defects in insulin signaling. Binding of exendin-4 to GLP1 receptors and of insulin to IRs prevented activation of JNK, allowing physiological tyrosine phosphorylation of IRS-1 and stimulating downstream insulin signaling. In both panels, red arrows indicate inhibitory pathways and green arrows indicate stimulatory pathways of insulin signaling. I, insulin.

Article Snippet: Antibodies against total IRS-1 and IRS-2, IRS-1pTyr465, IRS-1pSer636, glucagon-like peptide–1 receptor (GLP1R), TNFR1, TNF-α, and the PKR inhibitor were from Santa Cruz Biotechnology Inc. IRS-1pSer307, IRS-1pSer312, and IRS-1pSer616 antibodies were from Invitrogen.

Techniques: Activation Assay, Phospho-proteomics, Blocking Assay, Binding Assay

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: 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: eLife

Article Title: The switch-like expression of heme-regulated kinase 1 mediates neuronal proteostasis following proteasome inhibition

doi: 10.7554/eLife.52714

Figure Lengend Snippet:

Article Snippet: The PKR inhibitor ( ; ) (Cayman) was dissolved in DMSO and used at a final concentration of 1 μM and was added 1 hr before proteasome inhibitor treatment.

Techniques: Transfection, Construct, Ubiquitin Proteomics, Recombinant, Software, Generated, Sequencing, TaqMan 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: British Journal of Cancer

Article Title: Attenuation of muscle atrophy in a murine model of cachexia by inhibition of the dsRNA-dependent protein kinase

doi: 10.1038/sj.bjc.6603704

Figure Lengend Snippet: Effect of daily s.c. administration of a PKR inhibitor at 1 (▪) and 5 (▴) mg kg −1 in comparison with solvent control (DMSO : PBS 1 : 20) on body weight change ( A ) and tumour growth rate ( B ) in mice bearing the MAC16 tumour. A time course for the inhibition of body weight loss (▴) and tumour growth (▪) is shown in ( C ). The average weight of the soleus muscles after 5 days treatment is shown in ( D ), and the body composition is shown in ( E ). The conditions for tumour transplantation and conductance of the experiment are given in Materials and Methods. The number of mice in each group n =6. Differences from control are shown as a: P <0.05; b: P <0.01; or c: P <0.001, whereas differences from percentage inhibition of tumour volume are shown as f: P <0.001.

Article Snippet: The PKR inhibitor and PhosphoSafeTM Extraction Reagent were from Merck Eurolab Ltd (Leicestershire, UK) and electrophoretic mobility shift assay (EMSA) gel shift assay kits were from Panomics (CA, USA).

Techniques: Comparison, Solvent, Control, Inhibition, Muscles, Transplantation Assay

Journal: British Journal of Cancer

Article Title: Attenuation of muscle atrophy in a murine model of cachexia by inhibition of the dsRNA-dependent protein kinase

doi: 10.1038/sj.bjc.6603704

Figure Lengend Snippet: Western blots of phospho-PKR ( A ) and -eIF2 α ( B ) in gastrocnemius muscle of mice bearing the MAC16 tumour after 5 days treatment with a PKR inhibitor (1 and 5 mg kg −1 ), as described in the legend to . The blots for total PKR and eIF2 α were used as loading controls. The first lane (CON) used gastrocnemius muscle from an NTB control. Representative blots are shown and the densitometric analysis gives the ratio of the phospho to total forms as an average of three separate blots ( n =9). Differences from NTB control are shown as a: P <0.05 or c: P <0.001, whereas differences from the solvent control are indicated as f: P <0.01.

Article Snippet: The PKR inhibitor and PhosphoSafeTM Extraction Reagent were from Merck Eurolab Ltd (Leicestershire, UK) and electrophoretic mobility shift assay (EMSA) gel shift assay kits were from Panomics (CA, USA).

Techniques: Western Blot, Control, Solvent

Journal: British Journal of Cancer

Article Title: Attenuation of muscle atrophy in a murine model of cachexia by inhibition of the dsRNA-dependent protein kinase

doi: 10.1038/sj.bjc.6603704

Figure Lengend Snippet: Western blots of 20S proteasome α -subunits ( A ), p42 ( B ) and myosin ( C ) in gastrocnemius muscle of mice bearing the MAC16 tumour after 5 days treatment with a PKR inhibitor (1 and 5 mg kg −1 ), as described in the legend to in comparison with values from non-tumour-bearing animal (NTB). Actin was used as a loading control. The first lane (CON) used gastrocnemius muscle from a non-tumour-bearing control. Representative blots are shown and the densitometric analysis is the average of at least three separate blots. Differences from NTB control are shown as b: P <0.01 or c: P <0.001, whereas differences from the solvent control are indicated as f: P <0.001 ( n =9).

Article Snippet: The PKR inhibitor and PhosphoSafeTM Extraction Reagent were from Merck Eurolab Ltd (Leicestershire, UK) and electrophoretic mobility shift assay (EMSA) gel shift assay kits were from Panomics (CA, USA).

Techniques: Western Blot, Comparison, Control, Solvent

Journal: British Journal of Cancer

Article Title: Attenuation of muscle atrophy in a murine model of cachexia by inhibition of the dsRNA-dependent protein kinase

doi: 10.1038/sj.bjc.6603704

Figure Lengend Snippet: ( A ) EMSA of nuclear binding of NF- κ B in gastrocnemius muscle of non-tumour-bearing mice (lane 1) or mice bearing the MAC16 tumour and treated with solvent alone (lanes 2–4) or the PKR inhibitor at 1 (lanes 5–7) or 5 mg kg −1 (lanes 8–10). Lane 11 is a positive control for NF- κ B (supplied by the manufacturer of the kit). ( B ) Densitometric analysis of the EMSA shown in ( A ), n =3. Differences from non-tumour-bearing controls are shown as c: P <0.001, whereas differences from solvent control are shown as e: P <0.01 or f: P <0.001.

Article Snippet: The PKR inhibitor and PhosphoSafeTM Extraction Reagent were from Merck Eurolab Ltd (Leicestershire, UK) and electrophoretic mobility shift assay (EMSA) gel shift assay kits were from Panomics (CA, USA).

Techniques: Binding Assay, Solvent, Positive Control, Control

Journal: British Journal of Cancer

Article Title: Attenuation of muscle atrophy in a murine model of cachexia by inhibition of the dsRNA-dependent protein kinase

doi: 10.1038/sj.bjc.6603704

Figure Lengend Snippet: Flow diagram showing how activation of PKR leads to inhibition of protein synthesis through phosphorylation of eIF2 α ; and increased protein degradation through activation of NF- κ B, which would be attenuated by a PKR inhibitor (PKRI).

Article Snippet: The PKR inhibitor and PhosphoSafeTM Extraction Reagent were from Merck Eurolab Ltd (Leicestershire, UK) and electrophoretic mobility shift assay (EMSA) gel shift assay kits were from Panomics (CA, USA).

Techniques: Activation Assay, Inhibition, Phospho-proteomics

Journal: International Journal of Molecular Medicine

Article Title: Anti-β 2 GPI/β 2 GPI induces neutrophil pyroptosis and thereby enhances ICAM-1 and IL-8 expression in endothelial cells

doi: 10.3892/ijmm.2022.5120

Figure Lengend Snippet: Association between the PKR/p38MAPK signaling and anti-β 2 GPI/β 2 GPI complex-induced neutrophil pyroptosis. (A and B) Anti-β 2 GPI/β 2 GPI induced (A) PKR and (B) p38MAPK phosphorylation, which was respectively inhibited by TAK-242 and GC17925 treatment. (C) NLRP3, caspase-1 and pro-IL-1β protein levels were reduced by GC17925 or SB203580 treatment. (D) IL-1β secretion was decreased by GC17925 or SB203580 treatment. (E) GSDMD fluorescent staining demonstrated inhibition of anti-β 2 GPI/β 2 GPI-induced neutrophil pyroptosis mediated by GC17925 or SB203580 (scale bar, 50 μ m). Values are expressed as the mean ± standard error of the mean (n=3 and n=4 in D). * P<0.05, ** P<0.01, *** P<0.001. NLRP3, NLR family pyrin domain containing 3; anti-β2GPI, anti-β2-glycoprotein I; LPS, lipopolysaccharide; Ctrl, control; IC, anti-β 2 GPI/β 2 GPI immune complex; GSDMD, gasdermin D; p-/t-PKR, phosphorylated/total double-stranded RNA-dependent protein kinase.

Article Snippet: In certain experiments, neutrophils were pretreated for 30 min with 1 μ M of the TLR4 inhibitor TAK-242, 10 μ M of the PKR inhibitor GC17925 (GLPBIO), 10 μ M of the p38MAPK inhibitor SB203580 (MCE) or 1 μ M of the NLRP3 inhibitor MCC950 (MCE).

Techniques: Phospho-proteomics, Staining, Inhibition, Control

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