Journal: Archives of toxicology

Article Title: Cannabidivarin and cannabigerol induce unfolded protein response and angiogenesis dysregulation in placental trophoblast HTR-8/SVneo cells.

doi: 10.1007/s00204-024-03781-8

Figure Lengend Snippet: Fig. 2 CBDV and CBG increase the expression of ER-stress mark- ers. HTR-8/SVneo cells were treated with CBDV and CBG (5 μM) for 48 h, with or without the TRPV1 antagonist CPZ (0.2 µM). A-D Expression of the HSPA5, sXBP1, ATF4 and DDIT3 genes was analyzed through RT-PCR. Results show transcript levels normal- ized against ACTB. E The protein expression of p-eIF2α and CHOP

Article Snippet: Membranes were incubated with mouse monoclonal antibodies against CHOP (1:100, sc-7351; Santa Cruz Biotechnology, CA, USA) and TRB3 (1:100, sc-271572; Santa Cruz Biotechnology, CA, USA), or rabbit monoclonal or polyclonal antibodies against p-eIF2α (1:200, 3398S; Cell Signaling Technology, Leiden, The Netherlands), eIF2α (1:200, 5342S; Cell Signaling Technology, Leiden, The Netherlands), caspase-3 (1:200, sc-7148; Santa Cruz Biotechnology, CA, USA), poly (ADP-ribose) polymerase-1 (PARP-1) (1:200, 9542S; Cell Signaling Technology, Leiden, The Netherlands), p-AKT (Ser473) (1:200, 4060S; Cell Signaling Technology, Leiden, The Netherlands) and AKT (1:200, 4691S; Cell Signaling Technology, Leiden, The Netherlands) at 4 °C overnight.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Developmental biology

Article Title: Dephosphorylation of eIF2α is essential for protein synthesis increase and cell cycle progression after sea urchin fertilization.

doi: 10.1016/j.ydbio.2012.03.002

Figure Lengend Snippet: Fig. 2. eIF2α is dephosphorylated after fertilization. A—Eggs were fertilized and embryos were taken for Western blot analysis at indicated times after fertilization. Total extracts were analyzed by Western blot using an antibody directed against the Ser-51 phosphorylated form of eIF2α (P-eIF2α) and against the full length eIF2α protein (FL-eIF2α). B—The protein synthesis activity was measured by [35S]-L-methionine incor- poration (circles); eIF2α phosphorylation level was quantified on Western blots and expressed as a ratio of the phospho- to total eIF2α (squares). These data are representative of at least three independent experiments.

Article Snippet: Rabbit polyclonal antibody directed against phosphorylated Ser51 eIF2α (9721) was obtained from Cell Signaling.

Techniques: Western Blot, Activity Assay, Phospho-proteomics

Journal: Cell Cycle

Article Title: Evidence for eIF2α phosphorylation-independent effects of GSK2656157, a novel catalytic inhibitor of PERK with clinical implications

doi: 10.4161/cc.27726

Figure Lengend Snippet: Figure 1. Analysis of the effects of GSK2656157 on cells with conditionally active PERK or PKR. ( A ) Schematic representation of the GyrB system. The regulatory domain (RD) of either PKR (i.e., dsRNA-binding domain) or PERK (i.e., lumenal domain) was replaced by the GyrB domain, which was fused to the kinase domain (KD) of each eIF2α kinase. Coumermycin mediates the dimerization of the chimera kinase leading to its activation and induction of eIF2α phosphorylation. ( B and C ) GyrB.PERK cells were left untreated ( B , lane 1) or treated with 100 ng/ml coumermycin for 6 h in the absence ( B , lane 2; C , lane 1) or presence of the indicated concentrations of the PERK inhibitor (PERKi) ( C , lanes 2–6). ( D ) GyrB.PKR cells were left untreated (lane 1) or treated with 100 ng/ml coumermycin for 6 h in the absence (lanes 2 and 3) or presence of the indicated concentrations of PERKi (lanes 4–8). ( B–D ) Cells extracts (50 μg of protein) were subjected to immunoblot analyses for the indicated proteins.

Article Snippet: The antibodies used were: rabbit monoclonal against phosphorylated eIF2α at S51 (Novus Biologicals), mouse monoclonal anti-eIF2α, and rabbit polyclonal anti-PERK T980 antibodies (Cell Signaling Technology), mouse monoclonal antibody to PERK, mouse monoclonal antibody against actin (Clone C4, ICN Biomedicals Inc).

Techniques: Binding Assay, Activation Assay, Phospho-proteomics, Western Blot

Journal: Cell Cycle

Article Title: Evidence for eIF2α phosphorylation-independent effects of GSK2656157, a novel catalytic inhibitor of PERK with clinical implications

doi: 10.4161/cc.27726

Figure Lengend Snippet: Figure 2. Control of eIF2α phosphorylation by GSK2656157 in HT1080 cells. ( A ) Parental HT1080 cells were treated with the indicated concentrations of PERKi in the absence (−) or presence (+) of 1 μM thapsigargin (TG). As control untreated cells (lanes 1, 9, and 13) or cells treated 1 μM TG in the absence of PERKi (lanes 2, 10, and 14) were used. ( B ) Parental HT1080 cells were left untreated (lane 1) or treated with the indicated concentrations of the PERKi for 24 h (lanes 3–7). As control for eIF2α phosphorylation, cells were treated with 1 μM TG for 2 h (lane 2). ( A and B ) Cells extracts (50 μg of protein) were subjected to immunoblot analyses for the indicated proteins.

Article Snippet: The antibodies used were: rabbit monoclonal against phosphorylated eIF2α at S51 (Novus Biologicals), mouse monoclonal anti-eIF2α, and rabbit polyclonal anti-PERK T980 antibodies (Cell Signaling Technology), mouse monoclonal antibody to PERK, mouse monoclonal antibody against actin (Clone C4, ICN Biomedicals Inc).

Techniques: Control, Phospho-proteomics, Western Blot

Journal: Cell Cycle

Article Title: Evidence for eIF2α phosphorylation-independent effects of GSK2656157, a novel catalytic inhibitor of PERK with clinical implications

doi: 10.4161/cc.27726

Figure Lengend Snippet: Figure 4. Characterization of the biological properties of GSK2656157 in tumor cells exposed to ER stress. ( A ) Cell extracts (50 μg of protein) from wild-type (WT) and knock-in (KI) HT1080 cells untreated or treated with 1 μM thapsigargin (TG) for 2 h were subjected to western blot analysis for the indicated proteins. Note the delayed migration of the HA-eIF2αS51A in knock-in (KI) cells (lanes 3 and 4) compared with endogenous eIF2α in wild-type (WT) cells (lanes 1 and 2). ( B ) HT1080 WT and KI cells were treated with 1 μM TG for either 24 h. ( C ) Cells were treated with 1 μM TG either in the absence or presence of the indicated concentrations of PERKi for 18 h. As control, cells were treated with the same concentrations of PERKi in the absence of TG for 18 h. ( B and C ) Cell death was assessed by the percentage of cells in sub-G 1 population by FACS analysis. Histograms represent the quantification of 3 independent experiments.

Article Snippet: The antibodies used were: rabbit monoclonal against phosphorylated eIF2α at S51 (Novus Biologicals), mouse monoclonal anti-eIF2α, and rabbit polyclonal anti-PERK T980 antibodies (Cell Signaling Technology), mouse monoclonal antibody to PERK, mouse monoclonal antibody against actin (Clone C4, ICN Biomedicals Inc).

Techniques: Knock-In, Western Blot, Migration, Control

Journal: Veterinary research

Article Title: GADD34-mediated dephosphorylation of eIF2α facilitates pseudorabies virus replication by maintaining de novo protein synthesis.

doi: 10.1186/s13567-021-01018-5

Figure Lengend Snippet: Figure 2 PRV infection reduced the level of phosphorylated eIF2α in vitro and in vivo. PK15 cells infected with PRV at an A MOI of 0.1 or B MOI of 1.0 were harvested and lysed at 0, 3, 6, 9, 12, and 24 hpi. The levels of p-eIF2α, eIF2α, and PRV proteins were determined by Western blot analysis. The intensities of the p-eIF2α bands were determined by densitometry, normalized to eIF2α, and shown as fold changes (bottom panel). The values are presented as the mean ± SD of triplicate experiments. ** p < 0.01; *** p < 0.001. C BALB/c mice were intraperitoneally inoculated with PRV (1 × 106 PFU) and were then euthanized at 0, 24, 48, and 72 hpi. The levels of p-eIF2α, eIF2α, and PRV proteins in the lungs were determined by Western blot analysis. The intensities of the p-eIF2α bands were determined by densitometry, normalized to eIF2α, and shown as fold changes (right panel). The values are presented as the mean ± SD of triplicate experiments. * p < 0.05.

Article Snippet: The antibody against total eIF2α (sc-11386) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Infection, In Vitro, In Vivo, Western Blot

Journal: Veterinary research

Article Title: GADD34-mediated dephosphorylation of eIF2α facilitates pseudorabies virus replication by maintaining de novo protein synthesis.

doi: 10.1186/s13567-021-01018-5

Figure Lengend Snippet: Figure 3 Salubrinal increased the level of p-eIF2α and suppressed PRV replication in PK15 cells. A PK15 cells were infected with PRV at an MOI of approximately 0.1 in the presence of 100 μM salubrinal. The cells were subjected to puromycin labelling for 1 h at 23 hpi and were then harvested at 24 hpi. Cell lysates were subjected to Western blot analysis to determine the levels of p-eIF2α, eIF2α, and PRV proteins. The intensities of the p-eIF2α bands were determined by densitometry, normalized to eIF2α, and shown as fold changes (right panel). PRV protein expression was quantified by densitometry and normalized to β-actin and are shown as fold changes (right panel). The values are presented as the mean ± SD of triplicate experiments. * p < 0.05; ** p < 0.01. B De novo protein synthesis was measured by using a monoclonal antibody against puromycin; the intensities of bands corresponding to puromycin-labelled proteins were quantified by densitometry and normalized to β-actin and are shown as fold changes (right panel). The values are presented as the mean ± SD of triplicate experiments. * p < 0.05. C, D PK15 cells were infected with PRV at an MOI of approximately 0.1 in the presence of 100 μM salubrinal for 24 h, and supernatants and cells were then harvested. RT–qPCR was performed to determine the relative mRNA level of PRV-gE compared to β-actin (C); the viral titre in the supernatant was determined in PK15 cells (D). The data are presented as the mean ± SD of three independent experiments. *** p < 0.001.

Article Snippet: The antibody against total eIF2α (sc-11386) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Infection, Western Blot, Expressing, Quantitative RT-PCR

Journal: Veterinary research

Article Title: GADD34-mediated dephosphorylation of eIF2α facilitates pseudorabies virus replication by maintaining de novo protein synthesis.

doi: 10.1186/s13567-021-01018-5

Figure Lengend Snippet: Figure 4 Overexpression of eIF2αwt or eIF2α(S51D) in PK15 cells inhibited PRV replication. A PK15 cells were transfected separately with the pCMV-HA, eIF2αwt, eIF2α(S51D), and eIF2α(S51A) constructs for 24 h and were then infected with PRV. Cells were subjected to puromycin labelling for 1 h at 23 hpi and were then harvested at 24 hpi. Western blot analysis was performed to detect eIF2α and PRV proteins. PRV protein expression was quantified by densitometry and normalized to β-actin and are shown as fold changes (bottom panel). The values are presented as the mean ± SD of triplicate experiments. ** p < 0.01, *** p < 0.001. B, C PK15 cells were transfected separately with the pCMV-HA, eIF2αwt, eIF2α(S51D), or eIF2α(S51A) constructs for 24 h and were then infected with PRV for another 24 h. Supernatants and cells were harvested. RT–qPCR was performed to determine the relative mRNA level of PRV-gE compared to β-actin (B); the viral titre in the supernatants was determined in PK15 cells (C). The data are presented as the mean ± SD of three independent experiments. *** p < 0.001. D De novo protein synthesis was measured in pCMV-HA-, eIF2αwt-, eIF2α(S51D)-, and eIF2α(S51A)-transfected cells by using a monoclonal antibody against puromycin; the intensities of bands corresponding to puromycin-labelled proteins were quantified by densitometry and normalized to β-actin and are shown as fold changes (right panel). The values are presented as the mean ± SD of triplicate experiments. * p < 0.05. *** p < 0.001.

Article Snippet: The antibody against total eIF2α (sc-11386) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Over Expression, Transfection, Construct, Infection, Western Blot, Expressing, Quantitative RT-PCR

Journal: Veterinary research

Article Title: GADD34-mediated dephosphorylation of eIF2α facilitates pseudorabies virus replication by maintaining de novo protein synthesis.

doi: 10.1186/s13567-021-01018-5

Figure Lengend Snippet: Figure 6 Okadaic acid (OA) reduced PRV replication in PRV-infected PK15 cells. A PK15 cells were infected with PRV (MOI = 0.1) in cell culture medium containing 1 µM Tg and were then harvested at 24 hpi. Western blot analysis was performed to measure p-PERK, PERK, p-eIF2α, and eIF2α protein levels. B PK15 cells were infected with PRV (MOI = 0.1) and incubated at 37 °C for 8 h before the addition of OA at the indicated concentrations. Cells were then incubated for another 15 h in the presence of OA, followed by puromycin labelling for 1 h. Cells were harvested at 24 hpi. Western blot analysis was performed to measure p-eIF2α, eIF2α, and PRV protein levels. The intensities of the p-eIF2α bands were determined by densitometry, normalized to eIF2α, and shown as fold changes (bottom panel). The values are presented as the mean ± SD of triplicate experiments. ** p < 0.01; *** p < 0.001. C De novo protein synthesis and PRV protein expression were measured by Western blot analysis; the intensities of bands corresponding to puromycin-labelled proteins were quantified by densitometry and normalized to β-actin and are shown as fold changes. The intensities of the PRV protein bands were quantified by densitometry and normalized to β-actin and are shown as fold changes (right panels). The values are presented as the mean ± SD of triplicate experiments. * p < 0.05; ** p < 0.01; *** p < 0.001. D, E PK15 cells were infected with PRV (MOI = 0.1) and incubated at 37 °C for 8 h before the addition of OA at the indicated concentrations. Cells were then incubated for another 16 h. Supernatants and cells were harvested at 24 hpi. RT–qPCR was performed to determine the relative mRNA level of PRV-gE compared to β-actin (D); the viral titre in the supernatants was determined in PK15 cells (E). The data are presented as the mean ± SD of three independent experiments. ** p < 0.01; *** p < 0.001.

Article Snippet: The antibody against total eIF2α (sc-11386) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

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

Journal: Veterinary research

Article Title: GADD34-mediated dephosphorylation of eIF2α facilitates pseudorabies virus replication by maintaining de novo protein synthesis.

doi: 10.1186/s13567-021-01018-5

Figure Lengend Snippet: Figure 8 Knockdown of GADD34 in PK15 cells inhibited PRV replication. A PK15 cells were transfected with shNC or shGADD34 (#1, #2, #3, and #4) for 24 h and were then infected with PRV (MOI = 0.1). Cells were subjected to puromycin labelling for 1 h at 23 hpi and were then harvested at 24 hpi. Western blot analysis was performed to detect p-eIF2α, eIF2α, and PRV proteins; GADD34 and PRV protein expression was quantified by densitometry and normalized to β-actin and are shown as fold changes. The intensities of the p-eIF2α bands were quantified by densitometry, normalized to eIF2α, and shown as fold changes (bottom panels). The values are presented as the mean ± SD of triplicate experiments. * p < 0.05; ** p < 0.01; *** p < 0.001. B De novo protein synthesis was measured in GADD34 knockdown cells by using a monoclonal antibody against puromycin; the intensities of bands corresponding to puromycin-labelled proteins were quantified by densitometry and normalized to β-actin and are shown as fold changes (the lower panel). The values are presented as the mean ± SD of triplicate experiments. ** p < 0.01. C, D PK15 cells were transfected with shNC or shGADD34 (#1, #2, #3, and #4) for 24 h and were then infected with PRV (MOI = 0.1). Supernatants and cells were harvested at 24 hpi. RT–qPCR was performed to determine the relative mRNA level of PRV-gE compared to β-actin (C); the viral titre in the supernatants was determined in PK15 cells (D). The data are presented as the mean ± SD of three independent experiments. *** p < 0.001.

Article Snippet: The antibody against total eIF2α (sc-11386) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Knockdown, Transfection, Infection, Western Blot, Expressing, Quantitative RT-PCR

Journal: Molecular and Cellular Biology

Article Title: Phosphorylation of the Eukaryotic Translation Initiation Factor 4E-Transporter (4E-T) by c-Jun N-Terminal Kinase Promotes Stress-Dependent P-Body Assembly

doi: 10.1128/MCB.00544-12

Figure Lengend Snippet: 4E-T becomes phosphorylated in response to oxidative stress. (A) U2OS cells growing in serum were transfected with HA-tagged 4E-T. Subcellular localization of endogenous Dcp1 (P-body component, green) and HA-tagged 4E-T (red) was determined by indirect immunofluorescence. The colocalization of these factors appears yellow in the merged image. Nuclei were stained with DAPI (blue). (B) U2OS cells stably expressing HA-tagged 4E-T were grown in the presence of serum and treated for 45 min with arsenite (0.5 mM). Endogenous PABP (stress granule component, green) and HA-tagged 4E-T (red) were analyzed by immunofluorescence. Higher-magnification views of boxed areas are shown in insets at the bottom right and display 4E-T and Dcp1 colocalization (A) and the distinct localization of 4E-T and PABP (B). Bars, 10 μm. (C) HEK293 cells were treated with arsenite (0.5 mM) for 45 min and clotrimazole (mitochondrial stress inducer, 20 μM) or FCCP (metabolic stress inducer, 1 μM) for 60 min. The phosphorylation of 4E-T was assayed by mobility shift. Induction of oxidative stress by arsenite was verified by monitoring the phosphorylation of eIF2α on Ser51. (D) Endogenous 4E-T was immunoprecipitated (IP) from HEK293 cells treated with arsenite. Immunoprecipitates were treated with λ-phosphatase (λ-PPase), and 4E-T phosphorylation was assayed as described for panel C. (E) HEK293 cells were incubated for 45 min with the antioxidant agent N-acetyl-l-cysteine (NAC) at the indicated concentrations prior to arsenite treatment. 4E-T phosphorylation was assayed as described for panel C.

Article Snippet: Antibodies targeted against phospho-eIF2α, phospho-JNK (Thr183/Tyr185), phospho-p38 (Thr180/Tyr182), and poly(A)-binding protein (PABP) were purchased from Cell Signaling Technologies (Beverly, MA).

Techniques: Transfection, Immunofluorescence, Staining, Stable Transfection, Expressing, Phospho-proteomics, Mobility Shift, Immunoprecipitation, Incubation

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: dsRNA Binding Domain of PKR Is Proteolytically Released by Enterovirus A71 to Facilitate Viral Replication

doi: 10.3389/fcimb.2017.00284

Figure Lengend Snippet: EV-A71 infection results in the phosphorylation and cleavage of PKR. (A) Time course of cellular protein phosphorylation and protein levels after EV-A71 infection. RD cells were infected with EV-A71 at an m.o.i. of 10. At the indicated times post-infection, cell extracts were collected. The total cellular protein in the extracts was quantified, and equivalent amounts from each sample were subjected to western blot analysis for the detection of cellular PKR, phosphorylated-PKR (PKR-p), eIF2α, and phosphorylated eIF2α (eIF2α-p), as well as the expression of viral 3CD and 3C proteins. eIF2α expression was detected as a protein loading control. An asterisk marks non-specific bands. A representative result from three independent experiments is shown. (B) Cleavage of PKR after EV-A71 infection. RD cells were infected with EV-A71 at an m.o.i. of 10 or treated with STS for 2, 4, 6, 8, and 10 h. Cell extracts were collected at the indicated times for immunoblotting analysis to detect the cellular PKR and viral 3A protein levels. PARP cleavage was examined as an apoptosis marker. GAPDH expression was detected as a protein loading control. Asterisks indicate non-specific bands. A representative result from at least three reproducible experiments is shown.

Article Snippet: Mouse monoclonal antibody against green fluorescent protein (GFP) (SC-9996) and rabbit polyclonal antibodies against eIF2α (SC-11386) and poly (ADP-ribose) polymerase (PARP) (SC-7150) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Infection, Phospho-proteomics, Western Blot, Expressing, Control, Marker

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: dsRNA Binding Domain of PKR Is Proteolytically Released by Enterovirus A71 to Facilitate Viral Replication

doi: 10.3389/fcimb.2017.00284

Figure Lengend Snippet: EV-A71 3C associates with PKR and induces PKR phosphorylation. (A) The 293T cells were cotransfected with plasmids encoding GFP-PKR-K296H and 3C-Flag, or 3C mutant C147S-Flag, and then harvested at 24 h post-transfection. The cell lysates were immunoprecipitated with antibody against Flag. Samples were then subjected to western blot analysis with detection using anti-GFP and anti-Flag antibodies. * Denotes the heavy chains. (B) Mutations of the protease catalytic sites H40 and C147 of 3C caused loss of the PKR phosphorylation activity. RD cells were either transfected with 3C-Flag, H40D, C147S, H40D/C147S, or R84Q, for 24 h or infected with EV-A71/2231 at an m.o.i. of 10 for 8 h. Cellular extracts were collected and immunoblotting was performed for detecting PKR, PKR-p, eIF2α, eIF2α-p, CstF64, Flag, and viral 3C expression levels. CstF64 cleavage was used as a control for 3C catalytic activity. A representative result from three independent experiments is shown. (C) The PKR inhibitor 2-AP has no effect on 3C-induced apoptosis. The 3C- or H40D/C147S-transfected 293T cells were incubated with or without 2-AP for 24 h and then analyzed for apoptosis by flow cytometry using Annexin V and PI staining. The values shown in the lower left, lower right, and upper right quadrants of each panel represent the percentage of viable, apoptotic, and dead cells, respectively. Data are the means ± SD of values from three independent experiments.

Article Snippet: Mouse monoclonal antibody against green fluorescent protein (GFP) (SC-9996) and rabbit polyclonal antibodies against eIF2α (SC-11386) and poly (ADP-ribose) polymerase (PARP) (SC-7150) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Phospho-proteomics, Mutagenesis, Transfection, Immunoprecipitation, Western Blot, Activity Assay, Infection, Expressing, Control, Incubation, Flow Cytometry, Staining

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: dsRNA Binding Domain of PKR Is Proteolytically Released by Enterovirus A71 to Facilitate Viral Replication

doi: 10.3389/fcimb.2017.00284

Figure Lengend Snippet: Expression of a PKR kinase-dead mutant results in an increase of viral proteins and virus titer. (A) RD cells were transiently expressed with PKR or the K296H mutant for 24 h, and then infected with EV-A71 at an m.o.i. of 10. Immunoblot analysis was performed for detecting the presence and phosphorylation of PKR (anti-PKR, anti-PKR-p) and eIF2α (anti-eIF2α, anti-eIF2α-p), and the expression of viral 3A and 3C proteins. GAPDH expression was used as a protein loading control. A representative result from three independent experiments is shown. (B) Stable RD cells expressing vector alone, PKR, or K296H were selected by addition of 3 μg/mL puromycin. Cells were infected with EV-A71 at an m.o.i. of 10 and then the cellular extracts were harvested at 0, 6, and 8 h post-infection. Immunoblot analysis was performed to detect the presence of PKR and viral 3A and 3C proteins. A representative result based on three independent experiments is shown. (C) RD cells stably expressing PKR or the K296H mutant were infected with EV-A71 at an m.o.i. of 10 of for 8 h. The RD cells and culture supernatant were harvested for virus titer determination by plaque assay. The results are expressed as the mean ± SD ( n = 3). * p < 0.05.

Article Snippet: Mouse monoclonal antibody against green fluorescent protein (GFP) (SC-9996) and rabbit polyclonal antibodies against eIF2α (SC-11386) and poly (ADP-ribose) polymerase (PARP) (SC-7150) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Mutagenesis, Virus, Infection, Western Blot, Phospho-proteomics, Control, Plasmid Preparation, Stable Transfection, Plaque Assay

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: dsRNA Binding Domain of PKR Is Proteolytically Released by Enterovirus A71 to Facilitate Viral Replication

doi: 10.3389/fcimb.2017.00284

Figure Lengend Snippet: Proposed model of modulation of PKR function by EV-A71 3C protease. PKR consists of two dsRNA-binding motifs (dsRBM1 + dsRBM2 in green) and the C-terminal kinase domain (gray). In general, binding of viral dsRNA leads to dimerization and autophosphorylation of PKR. Active PKR subsequently phosphorylates its substrate eIF2α, which results in translation inhibition and apoptosis (left). Overexpression of the PKR-K296H mutant (cartoon molecule with a red x) competes with endogenous PKR for dsRNA binding to attenuate PKR activation (middle). In EV-A71 infection, 3C interacts with PKR, which may block its dimerization. Then, 3C cleaves PKR to release dsRNA-binding motifs, which may compete with PKR for the recognition of dsRNA, thereby attenuating PKR activation and increasing viral replication (right).

Article Snippet: Mouse monoclonal antibody against green fluorescent protein (GFP) (SC-9996) and rabbit polyclonal antibodies against eIF2α (SC-11386) and poly (ADP-ribose) polymerase (PARP) (SC-7150) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Binding Assay, Inhibition, Over Expression, Mutagenesis, Activation Assay, Infection, Blocking Assay

Journal: Cellular immunology

Article Title: Effects of cigarette smoke extract on primary activated T cells

doi: 10.1016/j.cellimm.2013.04.005

Figure Lengend Snippet: A. Phosphorylation of eIF2 was determined in stimulated T cells (A) or CCRF-CEM cells (B) cultured in the presence or the absence of CSE (5%). Densitometry analysis of band intensities (p-eIF2 /teIF2 ) was performed. C-D. CCRF-CEM cells expressing eIF2 -S51S or eIF2 -S51A were cultured in medium containing or not CSE (5%) and the expression of annexin V (C) or the levels of ATP (D) were determined. E. Culture of CCRF-CEM cells in CSE-medium in the presence of GSHe, but not catalase, prevented the induction of phospho-eIF2 after 2 hours of culture.

Article Snippet: Twenty micrograms of cell lysates, collected as described [ 20 ] were ran in 8% Tris-Glycine gels, transferred to PVDF membranes (Invitrogen), and immunobloted with specific antibodies against phospho-eIF2α (Epitomics, Burlingame, CA), total eIF2α (Invitrogen), and -actin (Sigma-Aldrich).

Techniques: Phospho-proteomics, Cell Culture, Expressing