Journal: Viruses

Article Title: Silvestrol Inhibits Chikungunya Virus Replication

doi: 10.3390/v10110592

Figure Lengend Snippet: Analysis of signal transduction during CHIKV infection in the presence of 50 nM silvestrol. ( A ) HEK 293T cells were seeded in six-well plates and were infected with CHIKV using an MOI of 1, and 50 nM silvestrol was added where indicated. Cells were treated with IFNα for 30 min before harvest if indicated, and Western blot analysis of cell lysates was performed. (−) Untreated cells; (+) treated cells. In lanes 3–6, cells were infected with CHIKV. The CHIKV E2 protein, STAT1, eIF2α, and their phosphorylated proteins were detected with specific antibodies and secondary HRP-coupled antibodies, and the ECL detection system (Amersham, Freiburg). Equal loading of each blot was controlled by detection of β-actin; and, ( B ) Uninfected HEK293T cells were treated with IFNα for 30 min before harvest and either treated with silvestrol for 16 h or left untreated. Western blot analysis of cell lysates was performed and p-STAT1 and STAT1 were detected. STAT1 served as a loading control.

Article Snippet: The following conditions were used: 50 mM sodium borate pH 9.0, 20% methanol, and 0.1% SDS buffer at 100 mA per membrane for 75 min. After the blotting, membranes were blocked with Roti-BlockTM (Carl Roth, Karlsruhe, Germany), and specific proteins were detected with antibodies directed against: CHIKV-E2 (Eurogentec, Köln, Germany, custom made), STAT1 (Sigma, Munich, Germany; #HPA000982), phospho-STAT1 (Cell Signaling, Frankfurt am Main, Germany; #7649), eIF2α (R&D Systems, Abingdon, UK; #AF3997), phospho-eIF2α (R&D Systems, Abingdon, UK; #MAB39971), mCherry (Abcam, Cambridge, UK, #ab183628), CHIKV-nsP2 (Abgenex, Bhubaneswar, India; clone ABM3F3.2E10), or β-actin (Sigma, Munich, Germany; #A5441).

Techniques: Transduction, Infection, Western Blot, Control

Journal: PLoS Pathogens

Article Title: Thriving under Stress: Selective Translation of HIV-1 Structural Protein mRNA during Vpr-Mediated Impairment of eIF4E Translation Activity

doi: 10.1371/journal.ppat.1002612

Figure Lengend Snippet: (A) HEK 293 cells were transfected with HIV-1 NL4-3 or ΔVifVprX (ΔVV) for 24 or 48 hour, as indicated. Equivalent whole-cell extracts were immunoblotted with antiserum against phospho-eIF2α Ser51, total eIF2α, phospho-eIF4E Ser209 or total eIF4E, respectively. (B) CEMx174 cells were infected with HIV-1 or ΔVifVprX (ΔVV) and evaluated at 12 hour intervals. Equivalent whole-cell extracts were immunoblotted with the indicated antiserum.

Article Snippet: The antibodies used for immunoblotting were: CBP80 (Bethyl A301–793A), eIF4E (Cell Signaling 9742), GRP78 (Abcam ab21685), α-tubulin (Santa Cruz sc-23948), HA.11 (Covance MMS-101R), HIV-1 Vif (6459, NIH AIDS reagent program), PARP (Cell Signaling 9542), β-Actin (Abcam ab6276), phospho-eIF2α Ser51 (Cell Signaling 3597), total eIF2α (Cell Signaling 9722), phospho-eIF4E Ser209 (Cell Signaling 9741), total eIF4E (Cell Signaling 9742), phospho-4E-BP1 Ser65 (Cell Signaling 9451), total 4E-BP1 (Cell Signaling 9452), phospho-Mnk1 Thr197/202 (Cell Signaling 2111), total Mnk1 (Cell Signaling 2195), Caspase-3 (Cell Signaling 9662) and CBP80 (Bethyl A301–793A).

Techniques: Transfection, Infection

Journal: PLoS Pathogens

Article Title: Thriving under Stress: Selective Translation of HIV-1 Structural Protein mRNA during Vpr-Mediated Impairment of eIF4E Translation Activity

doi: 10.1371/journal.ppat.1002612

Figure Lengend Snippet: (A) Serum deprivation or pharmacological cell cycle arrest is sufficient to reduce accumulation of phosphorylated eIF4E and 4E-BP1. HEK 293 cells were incubated in complete medium (serum +) or in low serum medium (−serum) for 24 hour. Equivalent whole-cell extracts were harvested and immunoblotted with phospho-eIF4E Ser209, total eIF4E, phospho-4E-BP1 Ser65, total 4E-BP1, phospho-Mnk1 Thr197/202, total Mnk1, phospho-eIF2α Ser51 and total eIF2α, antibodies respectively (left panel) . CEMx174 cells were incubated in complete medium that lacked or contained nocodazole (Noc) for 24 hour, and equivalent whole-cell extracts were immunoblotted with the indicated antiserum. (B) HEK 293 cells were transfected with expression plasmids encoding Vpr, indicated Vpr substitution mutant, or Vif, and whole-cell extracts were immunoblotted with the indicated antiserum.

Article Snippet: The antibodies used for immunoblotting were: CBP80 (Bethyl A301–793A), eIF4E (Cell Signaling 9742), GRP78 (Abcam ab21685), α-tubulin (Santa Cruz sc-23948), HA.11 (Covance MMS-101R), HIV-1 Vif (6459, NIH AIDS reagent program), PARP (Cell Signaling 9542), β-Actin (Abcam ab6276), phospho-eIF2α Ser51 (Cell Signaling 3597), total eIF2α (Cell Signaling 9722), phospho-eIF4E Ser209 (Cell Signaling 9741), total eIF4E (Cell Signaling 9742), phospho-4E-BP1 Ser65 (Cell Signaling 9451), total 4E-BP1 (Cell Signaling 9452), phospho-Mnk1 Thr197/202 (Cell Signaling 2111), total Mnk1 (Cell Signaling 2195), Caspase-3 (Cell Signaling 9662) and CBP80 (Bethyl A301–793A).

Techniques: Incubation, Transfection, Expressing, Mutagenesis

Journal: Nature Neuroscience

Article Title: Neuronal activity rapidly reprograms dendritic translation via eIF4G2:uORF binding

doi: 10.1038/s41593-024-01615-5

Figure Lengend Snippet: a , Immunofluorescence (IF) images of primary cortical neurons immunostained for glial fibrillary acidic protein (GFAP) and oligodendrocyte transcription factor 2 (OLIG2) simultaneously with PSD95 to show that the cultures are devoid of glial cells or oligodendrocytes, respectively. DAPI for nuclei; PSD95 for excitatory neurons. Magnification, ×40. Scale bars, 50 μm. b , TurboID-PSD95 was cloned without (top row) and with (bottom row) its 5′ and 3′ UTRs and lentivirally expressed in primary cortical neurons. White dashed boxes are zoomed in areas in black&white images. DAPI for nuclei; MAP2 for dendrites; Flag for each TurboID. % dendritically localized TurboID-PSD95 is quantified by co-localization with MAP2 signal in ImageJ. 3 different areas of images per replicate ( n = 3). Magnification, ×20. Scale bars, 50 μm. Significance was derived from biological replicates, showing the center line at mean. c , IF images of TurboID-PSD95-transduced neurons immunostained for DAPI (blue, for nuclei), PSD95 (red, for endogenous PSD95) and TurboID-PSD95 (cyan, detected by Flag). Magnification, ×60. Scale bar, 50 μm. d , IF images show the expression of a presynaptic marker, Synaptophysin (cyan), and TurboID-PSD95 (red, detected by Flag antibody) in primary cortical neurons transduced with TurboID-PSD95. DAPI (blue) marker for nuclei. Three zoomed in regions are marked by the white boxes. Magnification, ×60. Scale bar, 10 μm. e , IF images show TurboID expression and biotinylation in primary cortical neurons transduced with TurboID-PSD95 or Pan-TurboID after 30 minutes of biotin incubation. DAPI (blue, nuclei); MAP2 (green, dendrites); Flag (red, TurboID); and Streptavidin (cyan, biotinylated proteins). Magnification, ×20. Scale bars, 50 μm. f , Western blots stained for Flag and β-Actin from Pan-TurboID and TurboID-PSD95-transduced neurons in the absence (−) or presence (+) of exogenous biotin shown to indicate the relative expression levels of TurboID proteins. Quantifications of TurboID protein levels normalized to β-Actin are shown on the right ( n = 3); relative levels are not significant by two-tailed, paired Student’s t -test. g , Western blots stained for streptavidin signal in inputs (‘in’) and streptavidin pulldowns (‘pd’) from Pan-TurboID or TurboID-PSD95-transduced neurons in the absence (−) or presence (+) of exogenous biotin. h , Streptavidin pulldowns shown for dendritic (SHANK3, GKAP, NLGN1 and HOMER1) and negative control (GAPDH) proteins from TurboID-PSD95-transduced neurons in the absence (−) or presence (+) of exogenous biotin. Flag signal indicates self-biotinylation of each construct. Loaded on the gel are 10% (by volume) of input and 50% (by volume) of pulldowns. Percent isolated by TurboID-PSD95 in each condition is calculated by dividing the signal in the pulldown lane by that of the input lane, after each is adjusted to total, and quantifications are shown as bar graphs ( n = 3). P values: Flag = 0.58, SHANK3 = 0.0061, GKAP = 0.018, NLGN1 = 0.00052, HOMER1 = 0.021, GAPDH = 0.42. i , Streptavidin pulldowns shown for dendritic (BAIAP2 and DLGAP3) and nuclear (TBR1, H4 and H2AX) proteins from Pan-TurboID and TurboID-PSD95-transduced neurons in the presence (+) of exogenous biotin. Loaded on the gel are 10% (by volume) of input and 50% (by volume) of pulldowns. Percent isolated by each TurboID is calculated as in (h) ( n = 3). P values: BAIAP2 = 0.0052, DLGAP3 = 0.0035, TBR1 = 0.0063, H4 = 0.018, H2AX = 0.0037. j , Phosphorylation of EEF2, eIF2α, ERK1/2 and IRE1 and total levels of ATF4 and CHOP are shown in resting (rest), activated (DHPG, Dep) and stressed (Sodium arsenite (NaAsO 2 )) cells by using phospho-specific and total antibodies. The amount of phosphorylated or total protein is shown in the bar graphs, calculated by dividing the phosphorylated signal to total and β-Actin for the phosphorylated proteins and by dividing the total to β-Actin for ATF4 and CHOP ( n = 3). Significance was calculated with respect to rest. P values: P-EEF2 (DHPG = 0.0088, Dep = 0.0023, NaAsO 2 = 0.039), P-eIF2α (DHPG = 0.018, Dep = 0.0034, NaAsO 2 = 0.028), P-ERK1/2 (DHPG = 0.015, Dep = 0.0067, NaAsO 2 = 0.00084), P-IRE1 (DHPG = 0.06, Dep = 0.37, NaAsO 2 = 0.0027), ATF4 (DHPG = 0.038, Dep = 0.42, NaAsO 2 = 0.016), CHOP (DHPG = 0.044, Dep = 0.18, NaAsO 2 = 0.024). k , Quantitative PCR (qPCR) results shown for immediate early genes, Arc , Fos and Jun . The fold changes for each gene are calculated by first normalizing to the house-keeping gene β-Actin in each condition, then dividing the value of each condition by that of the resting state ( n = 3). l , Dendritic spine size in resting and KCl-depolarized neurons are measured using the Keyence microscope. Red squares are examples of spines that are counted ( n = 3, 12 spines from each biological replicate are counted as technical replicates). Significance was derived from the biological replicates using the two-tailed, unpaired Student’s t -test. Box plots show the min and max, with the center line at median. Magnification, ×100. Scale bars, 5 μm. m , Fluo-4-AM staining in resting, KCl-depolarized and DHPG-depolarized cells. Fluo4-AM was loaded in resting cells and measurements were taken at indicated time points after Fluo4-AM removal. In depolarized cells, the dye was loaded during silencing. After silencing, fluorescence was measured during stimulus at 10, 30 and 60-minute time points for the KCl treatment and at 10-minute for the DHPG-induced activation. Fluorescence was also measured 60 minutes after the stimulus removal (60′post KCl and 60′post DHPG). Circles represent data from 2 biological and 3 technical replicates. Below: Examples of Fluo4-AM fluorescence are shown in resting, 10-minute KCl-treated and 10-minute DHPG-treated neurons. Fluo4-AM loading (45 minutes) was performed during the last 45 minutes of the silencing step prior to stimulus addition for the KCl and DHPG treatment and simultaneously for the resting neurons. Imaging was performed 10 minutes after the stimulus was added. Scale bars, 50 μm. (b,f,h-k,m) Data are mean ± s.d. Significance was calculated using the two-tailed, paired Student’s t -test. P values: ns (not significant) >0.05; * <0.05; ** <0.01; *** <0.001; **** <0.0001. n indicates the number of biologically independent samples.

Article Snippet: Puromycin (1:3,000, mouse, Kerafast, EQ0001, RRID: AB_2620162), Flag (1:3,000, mouse, Sigma-Aldrich, F1804, RRID: AB_262044), β-Actin antibody (1:2,500, mouse, Sigma-Aldrich, A1978, RRID: AB_476692), RPL10A (1:1,000, rabbit, Abcam, ab174318), MAP2 (1:2,500, guinea pig, Synaptic Systems, 188004, RRID: AB_2138181), GFAP (1:500, rabbit, Abcam, ab7260, RRID: AB_305808), OLIG2 (1:500, rabbit, Proteintech, 13999-1-AP, RRID: AB_2157541), PSD95 (1:500, mouse, Millipore, MABN68, RRID: AB_10807979), Synaptophysin (1:300, mouse, Abcam, ab8049, RRID: AB_2198854), SHANK3 (1:500, mouse, Novus, NBP1-47610, RRID: AB_10010567), GKAP (1:500, rabbit, Novus, NBP1-76911, RRID: AB_11017331), NLGN1 (1:200, mouse, Novus, NBP2-42192), HOMER1 (1:1,000, rabbit, Proteintech, 12433-1-AP, RRID: AB_2295573), GAPDH (1:5,000, mouse, Thermo Fisher Scientific, AM4300, RRID: AB_2536381), BAIAP2 (1:500, rabbit, Proteintech, 11087-2-AP, RRID: AB_2063075), DLGAP3 (1:500, rabbit, Proteintech, 55056-1-AP, RRID: AB_10858793), TBR1 (1:500, rabbit, Proteintech, 20932-1-AP, RRID: AB_10695502), H4 (1:1,000, mouse, Abcam, ab31830, RRID: AB_1209246), H2A.X (1:1,000, rabbit, Proteintech, 10856-1-AP, RRID: AB_2114985), EEF2 (1:1,000, rabbit, Cell Signaling Technology, 2332, RRID:AB_10693546), P-EEF2 (1:1,000, rabbit, Cell Signaling Technology, 2331, RRID: AB_10015204), eIF2α (1:1,000, rabbit, Cell Signaling Technology, 9722, RRID: AB_2230924), P-eIF2α (1:1,000, rabbit, Cell Signaling Technology, 3398, RRID: AB_2096481), p42/44 MAPK (1:1,000, rabbit, Cell Signaling Technology, 4695, RRID: AB_390779), P-p42/44 MAPK (1:1,000, rabbit, Cell Signaling Technology, 9101, RRID: AB_331646), P-IRE1 (1:500, rabbit, Novus, NB100-2323SS, RRID: AB_10145203), IRE1 (1:500, rabbit, Novus, NB100-2324SS, RRID: AB_10000972), CHOP (1:1,000, mouse, Cell Signaling, 2895T, RRID: AB_2089254), ATF4 (1:1,000, rabbit, Cell Signaling Technology, 11815S, RRID: AB_2616025), MPHOSPH (1:300, rabbit, Biorbyt, orb100446), KCNJ9 (1:300, rabbit, LSBio, LS-C352416), KCNJ9 (1:300, mouse, Antibodies Incorporated, 75-445, RRID: AB_2686912), eIF4G2 (1:1,000, rabbit, Cell Signaling Technology, RRID: AB_10622189 and rabbit, Cell Signaling Technology, RRID: AB_2261993), NSUN3 (1:250, rabbit, LSBio, LS-C163024), MTF1 (1:300, rabbit, Novus, NBP1-86380, RRID: AB_11011361), ZFP64 (1:300, rabbit, Proteintech, 17187-1-AP, RRID: AB_2218826) and KATNBL1 (1:250, rabbit, Proteintech, 24795-1-AP, RRID: AB_2879730).

Techniques: Immunofluorescence, Clone Assay, Derivative Assay, Expressing, Marker, Transduction, Incubation, Western Blot, Staining, Two Tailed Test, Negative Control, Construct, Isolation, Real-time Polymerase Chain Reaction, Microscopy, Fluorescence, Activation Assay, Imaging

Journal: Methods (San Diego, Calif.)

Article Title: Methods for the characterization of stress granules in virus infected cells

doi: 10.1016/j.ymeth.2015.04.009

Figure Lengend Snippet: Commonly used antibodies for SG components.

Article Snippet: eIF2α total , Cell Signaling 2103S , Mouse , , Present in Pat A SGs, not arsenite SGs.

Techniques: Labeling, Marker

Journal: Circulation Research

Article Title: Carbon Monoxide Induces Heme Oxygenase-1 via Activation of Protein Kinase R–Like Endoplasmic Reticulum Kinase and Inhibits Endothelial Cell Apoptosis Triggered by Endoplasmic Reticulum Stress

doi: 10.1161/circresaha.107.154781

Figure Lengend Snippet: Figure 2. Effects of RuCO on PERK, eIF2, ATF4, HO-1, and Nrf2 in HUVECs. A, Cells were exposed to 20 mol/L RuCO, 10 mol/L TG, or 10 mol/L TM for indicated periods of time. B, Cells were exposed to 20 mol/L RuCO or 20 mol/L arsenite (positive control) for 160 minutes. C, Cells transfected with siRNA against eIF2 were exposed to RuCO for 1 hour. D, Cells were exposed for 1 hour (PERK and eIF2 phosphorylations) or 6 hours (ATF4 expression) to RuCO in the presence or absence of 50 g/mL Hb. E, Cells trans- fected with ether PERK siRNA or eIF2 siRNA were exposed to RuCO for 1 hour (Nrf2 nuclear translocation) or 6 hours (HO-1 expres- sion). NF indicates nuclear fraction. Western blot analysis and densitometry analysis were performed as described in Materials and Methods. Blots shown are representative of 3 independent experiments. Values are meansSD from 3 independent experiments. *P0.05 with respect to each untreated group.

Article Snippet: Predesigned siRNAs against human HO-1 (catalog no. SC-35554), PERK (catalog no. SC-36213), eIF2 (catalog no. SC-35272), CHOP (catalog no. SC-35437), and control scrambled siRNA (catalog no. SC-37007) were purchased from Santa Cruz Biotechnology.

Techniques: Positive Control, Transfection, Expressing, Translocation Assay, Western Blot

Journal: Circulation Research

Article Title: Carbon Monoxide Induces Heme Oxygenase-1 via Activation of Protein Kinase R–Like Endoplasmic Reticulum Kinase and Inhibits Endothelial Cell Apoptosis Triggered by Endoplasmic Reticulum Stress

doi: 10.1161/circresaha.107.154781

Figure Lengend Snippet: Figure 6. Effects of RuCO and RuCO-induced HO-1 on TG-, TM-, and HCys-induced CHOP in HUVECs. A, Cells were preincubated for 6 hours with 20 mol/L RuCO and then were exposed for 6 hours to 10 mol/L TG, 10 mol/L TM, or 50 mol/L HCys. B, Cells trans- fected with either PERK siRNA or eIF2 were preincubated for 6 hours without or with RuCO and then were exposed to TG for 6 hours. C, Normal cells and the cells transfected with siRNA against HO-1 were preincubated for 6 hours with RuCO or 10 mol/L CoPP in the presence or absence of 50 g/mL Hb and then exposed to TG for 6 hours. D, Normal cells and the cells transfected with PERK siRNA, HO-1 siRNA, or eIF2 siRNA were preincubated for 6 hours with or without RuCO and then were exposed to TG for 18 hours. Western blot analysis for CHOP, HO-1, ATF6, and Xbp-1 and MTT assay for cell viability were performed as described in Materi- als and Methods. Blots shown are representative of 3 independent experiments. Each bar represents meanSD from 3 independent experiments. *P0.05 with respect to each untreated group. *P0.05.

Article Snippet: Predesigned siRNAs against human HO-1 (catalog no. SC-35554), PERK (catalog no. SC-36213), eIF2 (catalog no. SC-35272), CHOP (catalog no. SC-35437), and control scrambled siRNA (catalog no. SC-37007) were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Western Blot, MTT 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: 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: Experimental hematology

Article Title: Panobinostat and venetoclax enhance the cytotoxicity of gemcitabine, busulfan, and melphalan in multiple myeloma cells

doi: 10.1016/j.exphem.2020.01.003

Figure Lengend Snippet: List of primary antibodies, their sources and dilutions

Article Snippet: eIF2α , Cell Signaling/9722 , Rabbit , 2500.

Techniques:

Journal: BioMed Research International

Article Title: Baicalein Induces Apoptosis and Autophagy via Endoplasmic Reticulum Stress in Hepatocellular Carcinoma Cells

doi: 10.1155/2014/732516

Figure Lengend Snippet: Diverse roles of UPR proteins in baicalein-induced apoptosis.(a) SMMC-7721 cells were transfected with scrambled RNA (NC) or CHOP-targeting siRNA (si-CHOP) for 48 h and treated with 0, 100, and 200 μ M baicalein for 24 h. Protein levels of cleaved PARP and CHOP were determined by western blotting. (b) SMMC-7721 cells were transfected with scrambled RNA (NC) or eIF2 α -targeting siRNA (si-eIF2 α ) and then treated with 0, 100, and 200 μ M baicalein for 24 h. Protein levels of cleaved PARP phosphorylated eIF2 α and eIF2 α were determined. (c) After being transfected with scrambled RNA (NC) or IRE1 α -targeting siRNA (si-IRE1 α ), SMMC-7721 cells were treated with the indicated dose of baicalein for 24 h and subjected to western blotting to analyze the level of cleaved PARP, IRE1 α , phosphorylated JNK, and total JNK. GAPDH served as a loading control.

Article Snippet: siRNAs against human eIF2 α , CHOP, IRE1 α , Beclin 1, and Atg5 were synthesized by GenePharma (Shanghai, China).

Techniques: Transfection, Western Blot, Control