Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Gestational long-term hypoxia induces metabolomic reprogramming and phenotypic transformations in fetal sheep pulmonary arteries

doi: 10.1152/ajplung.00469.2020

Figure Lengend Snippet: Effect of long-term hypoxia on sarcoplasmic endoplasmic reticulum stress in fetal lamb pulmonary arteries. Western blot analysis (A) was used to determine the ratio of phosphorylated protein/total protein of protein kinase RNA-like endoplasmic reticulum kinase (PERK) and eukaryotic initiation factor-2α (eIF2α) in pulmonary arteries of near-term fetal lambs from control and high-altitude hypoxic pregnant ewes (B). Data are means ± SD of three animals in each group. *P < 0.05 by an unpaired two-tailed t test, LTH vs. control. P-PERK, phospho-PERK; P-eIF2α, phospho-eIF2α.

Article Snippet: After blocking nonspecific binding sites by dry milk, membranes were incubated with primary antibodies (1:300 dilution) against phospho-PERK (bs-3330R, Bioss), PERK (bs-2469R, Bioss), phospho-eIF2α (bs-4842R, Bioss), or eIF2α (bs-3613R, Bioss).

Techniques: Western Blot, Two Tailed Test

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: G3: Genes|Genomes|Genetics

Article Title: Global Transcriptome Changes That Accompany Alterations in Serotonin Levels in Caenorhabditis elegans

doi: 10.1534/g3.120.401088

Figure Lengend Snippet: 5-HT treatment induced similar changes as seen in stressed animals. (A, B) Scatterplot showing the relationship between significantly enriched genes from 5-HT-treated animals and genes from a SPELL transcription dataset from the two enriched categories. Line represents linear regression, Pearson’s r value shown. p-value is corrected for multiple tests. (A) the relationship between significantly enriched genes from 5-HT-treated animals and animals in SPELL dataset belonging to the ‘Immune’ category. (B) the relationship between significantly enriched genes from 5-HT-treated animals and animals in the SPELL dataset belonging to the ‘Defense’ category. (C) Table showing the list of datasets and publications that showed a significant correlation with the 5-HT RNA-seq data. The left column indicates the Wombase ID and the right column the SPELL ID. (D) Phospho- eIF2α (p (Ser51)-eIF2α) levels in animals with elevated 5-HT. Top Panel shows representative Western Blot using anti-phospho (Ser51)-eIF2-α antibody. Lower Panel show Bar chart of the relative expression p(Ser51)-eIF2α normalized to the untreated control. Tubulin was used as a loading control. Bars in D represent mean, error bars correspond to standard error of the mean. Significance was determined using a student’s two-tailed t -test. P < 0.05.

Article Snippet: Rabbit anti-phospho (Ser51)-eIF2-α antibody (catalog no. 9721S, Cell Signaling Technology) was used to detect the level of phosphorylated eIF2-α.

Techniques: RNA Sequencing, Western Blot, Expressing, Control, Two Tailed Test

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:

Article Title: Alpha Interferon Induces Distinct Translational Control Programs To Suppress Hepatitis C Virus RNA Replication

doi: 10.1128/JVI.77.7.3898-3912.2003

Figure Lengend Snippet: Assessment of PKR activity and viral RNA levels within Huh7 control and HCV replicon cells. (A) Analysis of in vivo PKR activity during HCV RNA replication. The diagram shows comparative structural representations of the HCV genome (upper) and subgenomic replicon, denoting the HCV 5′-NTR/IRES, the EMCV IRES, and regions encoding the neomycin-resistance protein (Neo) and the various cleavage products of the HCV polyprotein. The panels show protein analyses of Huh7 control and replicon cells that were cultured alone (lanes 1 to 3) or in the presence of 40 μg of dsRNA/ml (lanes 4 to 6) as described in Materials and Methods. In the upper panel, the phosphorylation state (activity) of PKR was assessed by 32P metabolic labeling of proteins, followed by anti-PKR immunoprecipitation anal-ysis of control Huh7 cells (lanes 1 and 4) and Huh7 cells harboring the L2198S (lanes 2 and 5) or K2040 HCV replicon quasispecies (lanes 3 and 6). The lower panel is an immunoblot analysis of PKR levels present within identical parallel cultures of Huh7 control and replicon cells. The results shown are representative of three independent experiments. (B) HCV RNA levels in IFN-treated HCV replicon cells. HCV RNA levels were quantified by real-time RT-PCR assay of total RNA extracted from Huh7 cells harboring the L2198S or K2040 HCV replicon that were cultured for 24 h alone or with the indicated concentrations of IFN-α. Bars show combined data from three independent experiments presenting the overall average and standard deviation of RNA copy number relative to the GAPDH mRNA levels present within each sample. The percent viral RNA remaining after each 24 h IFN treatment is shown beneath the respective bar. (C) Protein expression and eIF2α phosphorylation in HCV replicon cell cultures. Huh7 control cells (lanes 1 and 2) and Huh7 cells harboring the K2040 (lanes 3 to 6) or L2198S HCV replicon quasispecies (lanes 7 to 10) were cultured alone or with 10 U of IFN-α/ml for the time indicated above each lane. Protein levels in cell extracts were determined by immunoblot analysis. The arrows point to the positions of the high-mass, hyperphosphorylated NS5A isoform or to P56. eIF2α-P denotes the expression of the S51-phosphorylated species of eIF2α. The relative levels of eIF2α-P from total eIF2α were quantified by densitometric analysis and are presented below each corresponding lane. The results shown are representative of three independent experiments.

Article Snippet: The primary antibodies used for immunoblot analysis were anti-HCV patient serum (obtained with informed consent from W. Lee), anti-PKR monoclonal antibody 71/10 (a kind gift from A. Hovanessian), rabbit polyclonal anti-phospho-eIF2α (Research Genetics, Inc.), anti-eIF2α monoclonal antibody ( 12 ), rabbit polyclonal anti-P56 antibody ( 19 ), goat polyclonal anti-actin antibody (Santa Cruz), and rabbit polyclonal anti-eIF3 antibody (a gift from J. Hershey) ( 3 ).

Techniques: Activity Assay, In Vivo, Cell Culture, Labeling, Immunoprecipitation, Western Blot, Quantitative RT-PCR, Standard Deviation, Expressing

Journal:

Article Title: Alpha Interferon Induces Distinct Translational Control Programs To Suppress Hepatitis C Virus RNA Replication

doi: 10.1128/JVI.77.7.3898-3912.2003

Figure Lengend Snippet: Influence of IFN and the PKR pathway upon HCV and EMCV IRES function. (A) Suppression of IRES function by IFN. Huh7 cells were transfected with the bicistronic plasmid reporter constructs pCMVRluc-EFluc or pRL-HL to simultaneously assess 5′ cap-dependent Renilla luciferase translation (cap) and EMCV IRES (upper panel) or HCV IRES-dependent firefly luciferase translation (middle panel), respectively. Cells were transfected and cultured for 24 h., followed by a 24-h incubation in medium alone or with medium containing increasing amounts of IFN-α. Luciferase activity, protein expression, and RNA levels were assessed from cell extracts. Each panel shows the luciferase values (an average from three experiments) derived from the 5′ cap (open bars) or viral IRES (shaded bars) as a percentage with standard deviations relative to cells cultured without IFN. The lower panel set shows Northern blot analyses of 3.3-kb pRL-HL bicistronic RNA (pRL-HL) and GAPDH (GDH) RNA levels (left) and a representative immunoblot analysis of PKR, P56, and actin protein levels (right) within the cultures corresponding to the pRL-HL transfected cells shown in the middle panel. Lanes show untransfected control cells (denoted as “C” on the Northern blot) or cells transfectedwith pRL-HL that were cultured for 24 h in the absence of IFN (lane 1) or increasing concentrations of IFN (lanes 2 to 4). We also conducted Northern blot analysis of RNA isolated from cells that were transfected with pCMVRluc-EFluc, and we confirmed that the corresponding bicistronic luciferase RNA was expressed to similar levels across all conditions (data not shown). (B) The PKR pathway influences IRES translation. Huh7 cells were cotransfected with bicistronic plasmid reporter constructs to simultaneously assess 5′ cap-dependent Renilla luciferase translation (cap) and EMCV IRES (upper panel) or HCV IRES-dependent firefly luciferase translation (middle panel) in the presence of an additional plasmid encoding the vector alone, the NS5A protein from the K2040 or L2198S HCV replicon, PKR K296R, or eIF2α S51A. At 24 h after transfection the culture medium was replaced with DMEM alone (IFN−) or DMEM containing 100 U of IFN-α/ml (IFN+) and, after an additional 24 h, the cells were harvested and extracts were subjected to the dual luciferase assay. Bars show the percentages of the luciferase levels relative to the values obtained from cultures cotransfected with the vector control (an average and standard deviation from three experiments) derived from 5′cap-dependent translation (open bars) or viral IRES-dependent translation (shaded bars). The lower panel set shows Northern blot analysis of the pRL-HL bicistronic luciferase RNA (pRL-HL) and GAPDH (GDH) RNA levels (left panel) and immunoblot analyses of NS5A, PKR, phospho-eIF2α (eIF2α-P), total eIF2α, and actin levels (right panel) in extracts derived from cells that were cotransfected with pRL-HL and expression constructs encoding the vector control (lanes 1 and 2), K2040 NS5A (lane 3), L2198S NS5A (lane 4), PKR K296R (lane 5; the hash mark denotes the position of HA-PKR K296R), or eIF2α S51A (lane 6), either treated or not treated with IFN as indicated. “C” (left panel set) denotes untransfected control cultures. In similar analyses, we confirmed that equal RNA and protein levels were present in cells cotransfected with pCMVRluc-Efluc (data not shown).

Article Snippet: The primary antibodies used for immunoblot analysis were anti-HCV patient serum (obtained with informed consent from W. Lee), anti-PKR monoclonal antibody 71/10 (a kind gift from A. Hovanessian), rabbit polyclonal anti-phospho-eIF2α (Research Genetics, Inc.), anti-eIF2α monoclonal antibody ( 12 ), rabbit polyclonal anti-P56 antibody ( 19 ), goat polyclonal anti-actin antibody (Santa Cruz), and rabbit polyclonal anti-eIF3 antibody (a gift from J. Hershey) ( 3 ).

Techniques: Transfection, Plasmid Preparation, Construct, Luciferase, Cell Culture, Incubation, Activity Assay, Expressing, Derivative Assay, Northern Blot, Western Blot, Isolation, Standard Deviation

Journal:

Article Title: Alpha Interferon Induces Distinct Translational Control Programs To Suppress Hepatitis C Virus RNA Replication

doi: 10.1128/JVI.77.7.3898-3912.2003

Figure Lengend Snippet: HCV RNA replication and IFN induce parallel translation control programs that impact virus replication. Our results demonstrate that HCV RNA replication has the capacity to activate PKR and to induce P56 expression through dsRNA signaling events that induce the host cell antiviral state (8, 40). During the antiviral response, PKR and P56 can function in parallel to limit viral RNA translation through the phosphorylation of eIF2α and disruption of eIF3 function, respectively. NS5A quasispecies that are competent to bind and inhibit PKR, such as the K2040 variant (40), can relieve the PKR-dependent translational control to increase the overall efficiency of viral RNA translation and replication. This regulation results in higher viral loads and may contribute a level of resistance against the antiviral actions of IFN (24). The HCV IRES, perhaps through a unique dependence upon eIF3 and/or the p48 eIF3 subunit (39), is acutely sensitive to the actions of P56. Our results suggest that sensitivity to P56 contributes to the dominant antiviral effects of IFN upon HCV IRES function, which may effectively limit HCV replication.

Article Snippet: The primary antibodies used for immunoblot analysis were anti-HCV patient serum (obtained with informed consent from W. Lee), anti-PKR monoclonal antibody 71/10 (a kind gift from A. Hovanessian), rabbit polyclonal anti-phospho-eIF2α (Research Genetics, Inc.), anti-eIF2α monoclonal antibody ( 12 ), rabbit polyclonal anti-P56 antibody ( 19 ), goat polyclonal anti-actin antibody (Santa Cruz), and rabbit polyclonal anti-eIF3 antibody (a gift from J. Hershey) ( 3 ).

Techniques: Expressing, Variant 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: bioRxiv

Article Title: The Integrated Stress Response Suppresses PINK1-dependent Mitophagy by Preserving Mitochondrial Import Efficiency

doi: 10.1101/2024.10.16.617214

Figure Lengend Snippet: (a) HEK293T PRKN OE LL cells expressing non-targeting control sgRNA ( NTC ), sgRNA targeting OMA1 ( OMA1 KD ) or HRI ( HRI KD) were left untreated or treated with 1.25 ng/mL oligomycin for 24 hr, followed by measurement of mitophagy via flow cytometry. (mean ± s.d., n = 3 culture wells) (b) Immunoblots of ATF4, HSPD1, PINK1, CoxIV, LC3, and β-Actin. NTC and HRI KD cells were left untreated or treated with 10 μM CCCP or 1.25 ng/mL oligomycin for 24 hr. β-Actin serves as loading control. (c) Wild type ( WT ) or two clonal eIF2α S49/52/A cells with PRKN OE HL were left untreated or treated with 1.25 ng/mL oligomycin with or without bafilomycin A (Baf), followed by measurement of mitophagy via flow cytometry. (mean ± s.d., n = 3 culture wells) (d) Immunoblots of ATF4, HSPD1, PINK1, CoxIV, LC3, and β-Actin in WT and eIF2α S49/52/A cells following 1.25 ng/mL oligomycin for 24 hr. β-Actin serves as the loading control. (e) Flow cytometry measurement of mitophagy in WT or two ATF4 KO clonal cell lines with PRKN OE HL following treatment with 1.25 ng/mL oligomycin for 24 hr in the presence or absence of bafilomycin A (Baf). (mean ± s.d., n = 3 culture wells) (f) Immunoblots of ATF4, HSPD1, PINK1, CoxIV and LC3 and β-Actin in WT and two ATF4 KO clonal cell lines following 10 μM CCCP or 1.25 ng/mL oligomycin treatment for 24 hr. β-Actin serves as the loading control. (g) NTC and DELE1 KD cells with PRKN OE HL were treated with 10 μM CCCP or 1.25 ng/mL oligomycin in the presence of cycloheximide at 12 different concentrations (0, 50 ng/mL, 100 ng/mL, 200 ng/mL, 400 ng/mL, 800 ng/mL, 1 μg/mL, 2 μg/mL, 4 μg/mL, 8 μg/mL,10 μg/mL and 20 μg/mL) for 24 hr followed by flow cytometry to measure mitophagy. Cycloheximide concentrations were converted to their base-10 logarithmic values. A nonlinear regression analysis using a log(inhibitor) vs. response model with a variable slope (four parameters) was performed to generate the plot. (mean ± s.d., n = 3 culture wells) (h) Immunoblots of ATF4, HSPD1, PINK1, CoxIV and LC3 and β-Actin in WT and DELE1 KD cells with PRKN OE HL following 10 μM CCCP or 1.25 ng/mL oligomycin treatment for 24 hr with or without 100 ng/mL cycloheximide (CHX). p: precursor; m: mature. β-Actin serves as the loading control. (i) NTC and DELE1 KD cells with PRKN OE HL are treated with 10 μM CCCP or 1.25 ng/mL oligomycin in the presence of torin1 at 7 different concentrations (0, 50, 100, 200, 250, 500 and 1000 nM) for 24 hr followed by flow cytometry to measure mitophagy. Torin1 concentrations were converted to their base-10 logarithmic values. A nonlinear regression analysis using a log(inhibitor) vs. response model with a variable slope (four parameters) was performed to generate the plot. (mean ± s.d., n = 3 culture wells) (j) Immunoblots of ATF4, HSPD1, PINK1, CoxIV and LC3 and β-Actin in NTC and DELE1 KD cells with PRKN OE HL following 10 μM CCCP or 1.25 ng/mL oligomycin treatment for 24 hr with or without 250 nM torin1. p: precursor; m: mature. β-Actin serves as the loading control.

Article Snippet: Antibodies used in this study include: anti-ATF4 (ProteinTech, 28657-1-AP, rabbit, 1:1000), anti-β-actin (ProteinTech, 66009-1-Ig, mouse, 1:5000), anti-β-actin (ProteinTech, 81115-1-RR, rabbit, 1:5000), anti-COXIV (Invitrogen, MA5-17279, mouse, 1:2000), anti-eIF2α (ProteinTech, 11170-1-AP, Rabbit, 1:1000), anti-GFP (Roche, 11814460001, mouse, 1:1,000), anti-HSPD1 (Invitrogen, MA3-012, mouse, 1:2000), anti-LC3 (ProteinTech, 14600-1-AP, Rabbit, 1:500), anti- phospho-eIF2α(ser51) (Cell Signaling Technology, 3398S, Rabbit, 1:1000), anti-PINK1 (ProteinTech, 23274-1-AP, rabbit, 1:500), anti-puromycin (Millipore Sigma, MABE343, mouse, 1:20000), anti-VDAC (Cell Signaling Technology, 4661S, Rabbit, 1:1000)

Techniques: Expressing, Control, Flow Cytometry, Western Blot