pkr inhibitor  (Santa Cruz Biotechnology)

Journal: Frontiers in Immunology

Article Title: Regulation of interferon alpha production by the MAGUK-family protein CASK under H5N1 infection

doi: 10.3389/fimmu.2024.1513713

Figure Lengend Snippet: Phosphorylated CASK enters nucleus and forms a complex to facilitate CRM1-mediated Ifna mRNP export during H5N1 infection When the H5N1 virus infects macrophages, PKR senses the 5’ tri-phosphate group of the viral genomic RNA, activating a signaling pathway that induces the expression of IFN-α. PKR activity is crucial for the transcriptional activation of Ifna , as treatment with a PKR inhibitor suppresses both CASK and IFN-α expression. During H5N1 infection, the expression and activity of HCK are upregulated, positively correlating with CDK5-mediated phosphorylation of CASK at S395. The S395-phosphorylated CASK translocates to the nucleus, where it forms a complex with STIP1, CCT4, and TNK1, as confirmed by IP-LC-MS/MS analysis. This CASK complex is recruited to the Ifna promoters through CCT4’s interaction with IRF7. Additionally, the complex associates with nascent Ifna mRNA via secondary interactions with HNRNPs and facilitates CRM1 recruitment to the transcript, enabling CRM1-mediated nuclear export. This Ifna mRNA-coupled CASK complex may counteract the competitive binding of influenza vRNP to CRM1, thereby supporting efficient nuclear export of Ifna mRNA.

Article Snippet: PKR inhibitor was purchased from Santa Cruz (sc-204200) and SFK inhibitor (PP2) was purchased from sigma (529573).

Techniques: Infection, Virus, Expressing, Activity Assay, Activation Assay, Phospho-proteomics, Liquid Chromatography with Mass Spectroscopy, Binding Assay

Journal: Frontiers in Neuroscience

Article Title: PKR downregulation prevents copper-induced synaptic dysfunction and cognitive impairment in a murine model of Wilson’s disease

doi: 10.3389/fnins.2024.1447304

Figure Lengend Snippet: Inhibiting the activation of the PKR/eIF2α pathway attenuates the synaptic dysfunction caused by copper in murine model of Wilson’s disease (WD). By preventing the phosphorylation of PKR in WD, the PKR inhibitor C16 effectively inhibits the PKR/eIF2α pathway and attenuates synaptic dysfunction, safeguarding against neuronal damage, cognitive decline, and neurodegenerative disorders.

Article Snippet: To inhibit PKR phosphorylation, we administered the PKR inhibitor PKR-IN-C16 (C16, Imidazolo-oxindole PKR inhibitor C16) (Selleck, USA) to WT or TX mice via intraperitoneal injections every morning for 30 days at a dose of 0.2 mL/mouse (300 μg/kg).

Techniques: Activation Assay, Phospho-proteomics

Journal: Glia

Article Title: Protein kinase R induced by type I interferons is a main regulator of reactive microglia in Zika virus infection

doi: 10.1002/glia.24619

Figure Lengend Snippet: In vivo, kinase PKR regulates the pro‐inflammatory and Aif1 gene expression response to ZIKV infection. CC071 mice (5–6 weeks‐old) were treated with DMSO or the inhibitor of PKR (IPKR) 1 h before and 3 days after IC inoculation of either NaCl (NI) or 10 5 FFU of ZIKV. Mice were necropsied 6 days after infection with one hemisphere used for immunofluorescence and the other one used for RNA extraction and gene expression analysis. (a–d) Levels of RNAs purified from whole brain extracts were determined by RT‐qPCR with respect to Hrpt1 used as reference gene. Symbols represent individual mice with n = 10 (NI + iPKR), n = 12 (ZIKV + DMSO) and n = 11 (ZIKV + iPKR). Dot plots show means with one dot for each brain and significance assessed by one‐way ANOVA Tukey's multiple comparison test. p ‐value <0.0001 (****), <0.001 (***), <0.01 (**), <0.05 (*) and ns = not significant; p ‐values near significance are indicated. (e) Correlation analysis of the expression levels of the Il1b , C4 , and Aif1 genes (as determined in NI + iPKR and ZIKV + DMSO conditions) with either Eif2ak2 expression or ZIKV RNA expression. Symbols represent individual mice. (f) Brain sections of ZIKV‐infected mice treated either with DMSO (ZIKV + DMSO) or PKR inhibitor C16 (ZIKV + iPKR) were labeled with anti‐NeuN (specific of neurons), anti‐IBA1 (specific of microglia) and anti‐NS2B (specific of ZIKV) antibodies and with DNA labeled with DAPI. Merge images of maximum intensity projection of confocal sections (z projection) are shown. Scale bars = 10 μm.

Article Snippet: When indicated, 20 μg of the PKR inhibitor C16 (Merck, 527450) were administered 45 min before and 3 days after infection.

Techniques: In Vivo, Expressing, Infection, Immunofluorescence, RNA Extraction, Purification, Quantitative RT-PCR, Comparison, RNA Expression, Labeling

Journal: Glia

Article Title: Protein kinase R induced by type I interferons is a main regulator of reactive microglia in Zika virus infection

doi: 10.1002/glia.24619

Figure Lengend Snippet: Kinase PKR is a main regulator of the phosphorylation of STAT1 and of IRF1 expression in microglial cells. (a–h, j) Primary cultured microglial cells (PCMCs) were incubated with DMSO or the inhibitor of PKR (C16) for 1 h before being (a, e) either non‐ (NI) or NDV‐infected and (c, g, j) either non‐treated (NT) or treated with conditioned media from non‐ (CM_NI) or ZIKV‐infected (CM_ZIKV) primary cultured neurons. Total protein extracts were collected 5 h after NDV infection and 6 h after treatment with CMs and submitted to a Western blot analysis. (b, d, f, h) Dot plots show means with one dot for each independent experiment represented in different colors. Significance was assessed by one‐way ANOVA Tukey's multiple comparison test. p ‐value <0.0001 (****), <0.05 (*), and ns = not significant. (i–k) Primary cultured neurons (PCNs) and microglial cells (PCMCs) were (i, j) either non‐ (NI) or NDV‐infected for 5 h or (k) treated with recombinant IFNB for 6 h. (i, j) Protein extracts or (k) RNAs were collected and submitted to Western blot or qPCR analysis respectively. (l) The level of expression of the Irf1 and Irf7 genes in total RNAs purified from whole brain extracts of mice described in Figure was analyzed by RT‐qPCR with respect to Hrpt1 used as reference gene. Symbols represent individual mice with n = 10 (NI + iPKR), n = 12 (ZIKV + DMSO), and n = 11 (ZIKV + iPKR). Dot plots show means with significance assessed by one‐way ANOVA Tukey's multiple comparison test. p ‐value <0.0001 (****) and ns = not significant.

Article Snippet: When indicated, 20 μg of the PKR inhibitor C16 (Merck, 527450) were administered 45 min before and 3 days after infection.

Techniques: Expressing, Cell Culture, Incubation, Infection, Western Blot, Comparison, Recombinant, Purification, Quantitative RT-PCR

Journal: Glia

Article Title: Protein kinase R induced by type I interferons is a main regulator of reactive microglia in Zika virus infection

doi: 10.1002/glia.24619

Figure Lengend Snippet: Kinase PKR is a major regulator of non‐infected microglia's inflammatory and phagocytic response to ZIKV‐infected neurons. Primary cultured microglial cells (PCMCs) were incubated with DMSO or the inhibitor of PKR (C16) for 1 h before being either non‐treated (NT), treated with conditioned media from non‐ (CM_NI) or ZIKV‐infected (CM_ZIKV) primary cultured neurons or ZIKV alone (NT_ZIKV) for 6 h. In Figure are shown the qPCR results obtained from three independent primary cultures of microglia treated with conditioned media collected from three independent primary cultures of neurons. RNAs were collected and gene expression analysis was carried out by RT‐qPCR with respect to Rplp0 used as reference gene for genes associated with (a) the pro‐inflammatory response, (b) IFN‐I response with the level of Ifnb1 expression present in the three ZIKV‐infected PCNs from which the CMs were collected (PCN_ZIKV 64 h) also shown, (c) DAM response. Dot plots show means with one dot for each independent experiment represented in different colors. Significance was assessed by ratio‐paired t ‐test. (d) After 15 min incubation with C3_SRBCs, phagocytosis index was determined as in Figure . Dot plots show means with one dot for each independent experiment. Significance was assessed by two‐way ANOVA Tukey's multiple comparison test. p ‐value <0.01 (**), <0.05 (*), and ns = not significant; p ‐values near significance are indicated.

Article Snippet: When indicated, 20 μg of the PKR inhibitor C16 (Merck, 527450) were administered 45 min before and 3 days after infection.

Techniques: Infection, Cell Culture, Incubation, Expressing, Quantitative RT-PCR, Comparison