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: 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

Journal: bioRxiv

Article Title: Harnessing the Evolution of Proteostasis Networks to Reverse Cognitive Dysfunction

doi: 10.1101/2025.02.28.640897

Figure Lengend Snippet: The Ppp1r15b R658C variant leads to persistent ISR activation and disrupt protein synthesis in the brain of Ppp1r15b R658C mice. (A) Sanger sequencing confirms the generation of Ppp1r15b R658C mice carrying the Ppp1r15b R658C variant. (B) Comparison of brain size between WT ( n = 6) and Ppp1r15b R658C ( n = 6) mice ( t = 7.82, P < 0.0001; two-tailed Student’s t -test). (C) Representative Western blot (top) and quantification (bottom) of PPP1R15B protein levels in hippocampal extracts from WT ( n = 4) and Ppp1r15b R658C mice ( n = 4) [ t = 1.42, P = 0.21]. (D) Immunoprecipitation of WT PPP1R15B and mutant PPP1R15B R658C using an GFP antibody followed by Western blots of endogenous PP1 and eIF2 (left). A schematic of the WT and mutant phosphatase complex (right). (E) Representative Western blot (top) and quantification (bottom) of eIF2-P levels in hippocampal extracts from WT ( n = 4) and Ppp1r15b R658C mice ( n = 5) [ t = 3.82 , P < 0.001]. (F) Schematic of polysome profiling sedimentation. After ultracentrifugation, sub-polysomes (40S, 60S, and 80S) and polysomes are separated based on their size. ( G-H ) Averaged polysome profile traces ( G ) and quantification ( H ) of polysome/sub-polysome ratio in the brain of WT and Ppp1r15b R658C mice ( n = 6 per group, t = 3.19 , P < 0.01). ( I-J ) Detection of puromycin incorporation into nascent peptides using an anti-puromycin antibody. A representative Western blot ( I ) and quantification ( J ) in hippocampal extract from WT ( n = 4) and Ppp1r15b R658C mice ( n = 3) [ t = 4.73, P < 0.01]. Data are mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: Antibodies used in this study: 119A11 eIF2-P (Cell Signaling Technology, Danvers, MA) or E90 ab32157 eIF2-P (Abcam, Cambridge, UK), D7D3 XP eIF2-T (Cell Signaling Technology, Dnvers, MA), M0802 GFP (Abiocode, Agoura Hills, CA), or A11192 GFP (Thermo Fisher Scientific, Waltham, MA), MABE343 puromycin (MilliporeSigma, Burlington, MA), sc7482 clone E9 PP1α (Santa Cruz Biotechnology, Santa Cruz, CA), PA5-31177 eIF2α (Thermo Fisher Scientific, Waltham, MA), A301-742A eIF2α (Bethyl Laboratories, Montgomery, TX), A300-721A eIF2α (Bethyl Laboratories, Montgomery, TX), 10449-1-AP PPP1R15A (Proteintech Group, Rosemont, IL), 14634-1- AP PPP1R15B (Proteintech Group, Rosemont, IL), 60004-1-IG GAPDH (Proteintech Group, Rosemont, IL).

Techniques: Variant Assay, Activation Assay, Sequencing, Comparison, Two Tailed Test, Western Blot, Immunoprecipitation, Mutagenesis, Sedimentation

Journal: bioRxiv

Article Title: Harnessing the Evolution of Proteostasis Networks to Reverse Cognitive Dysfunction

doi: 10.1101/2025.02.28.640897

Figure Lengend Snippet: Persistent ISR activation cause deficits in long-term synaptic plasticity and memory in Ppp1r15b R658C mice. (A) Schematic of the contextual fear conditioning paradigm. (B) Long-term contextual fear memory in WT ( n = 23) and Ppp1r15b R658C ( n = 18) mice ( F 3,52 = 60.76, t = 5.73, P < 0.0001). (C) L-LTP induced by four trains of high frequency stimulation (HFS, 4 x 100 Hz) in WT ( n = 8) and Ppp1r15b R658C ( n = 12) mice (at 250 min: t = 4.14, P < 0.001). (D) Representative Western blot (top) and quantification (bottom) of eIF2-P levels in hippocampal extracts from WT ( n = 7), Ppp1r15b R658C mice ( n = 9) and Ppp1r15b R658C -Eif2s1 S/A ( n = 3) [ F 2,16 = 1.86, WT vs. Ppp1r15b R658C : t = 4.25, P < 0.01; Ppp1r15b R658C vs. Ppp1r15b R658C -Eif2s1 S/A : t = 3.60, P < 0.01; WT vs. Ppp1r15b R658C - Eif2s1 S/A : t = 0.39, P > 0.99). (E) Long-term contextual fear memory in WT ( n = 7), Ppp1r15b R658C ( n = 13) and Ppp1r15b R658C - Eif2s1 S/A mice ( n = 8) [ F 2,50 = 5.58; WT vs. Ppp1r15b R658C : t = 4.31 , P < 0.001 ; Ppp1r15b R658C vs. Ppp1r15b R658C - Eif2s1 S/A : t = 5.12 , P < 0.0001; WT vs. Ppp1r15b R658C - Eif2s1 S/A : t = 0.54 , P > 0.99). (F) L-LTP induced by four trains of high frequency stimulation (HFS, 4 x 100 Hz) in WT ( n = 9) and Ppp1r15b R658C ( n = 5) mice and Ppp1r15b R658C - Eif2s1 S/A ( n = 8) [at 250 min: F 2,19 = 8.69; WT vs. Ppp1r15b R658C : t = 3.18 , P < 0.01; Ppp1r15b R658C vs. Ppp1r15b R658C - Eif2s1 S/A : t = 4.09, P < 0.001; WT vs. Ppp1r15b R658C - Eif2s1 S/A : t = 1.15, P = 0.264). Data are mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: Antibodies used in this study: 119A11 eIF2-P (Cell Signaling Technology, Danvers, MA) or E90 ab32157 eIF2-P (Abcam, Cambridge, UK), D7D3 XP eIF2-T (Cell Signaling Technology, Dnvers, MA), M0802 GFP (Abiocode, Agoura Hills, CA), or A11192 GFP (Thermo Fisher Scientific, Waltham, MA), MABE343 puromycin (MilliporeSigma, Burlington, MA), sc7482 clone E9 PP1α (Santa Cruz Biotechnology, Santa Cruz, CA), PA5-31177 eIF2α (Thermo Fisher Scientific, Waltham, MA), A301-742A eIF2α (Bethyl Laboratories, Montgomery, TX), A300-721A eIF2α (Bethyl Laboratories, Montgomery, TX), 10449-1-AP PPP1R15A (Proteintech Group, Rosemont, IL), 14634-1- AP PPP1R15B (Proteintech Group, Rosemont, IL), 60004-1-IG GAPDH (Proteintech Group, Rosemont, IL).

Techniques: Activation Assay, Western Blot

Journal: bioRxiv

Article Title: Harnessing the Evolution of Proteostasis Networks to Reverse Cognitive Dysfunction

doi: 10.1101/2025.02.28.640897

Figure Lengend Snippet: Protein alignment across diverse kingdoms of life reveals DP71L as a potent pan-ISR inhibitor. (A) Schematic of protein alignment showing PP1- and eIF2-binding motifs across various species. Consensus PP1- and eIF2-binding motif sequences are shown. (B) Overview of ISR kinases and the mechanism by which DP71L represses the ISR. ( C-E ) Cells expressing either GFP or GFP-DP71L were treated with the specific chemical inducer for each ISR kinase. Representative Western blot (top) and quantification (bottom) of eIF2-P levels upon activation of HRI ( C , F 3,16 = 7.14, n = 5 per group; GFP+Vehicle vs. GFP+Oligo: t = 4.22, P < 0.01; GFP-DP71L+Vehicle vs. GFP-DP71L+Oligo: t = 0.98, P > 0.99; GFP+Oligo vs. GFP-DP71L+Oligo: t = 4.17, P < 0.01), PKR [ D , n = 3-4 per group, F 3,10 = 9.80; GFP+Vehicle vs. GFP+Poly(I:C): t = 4.16, P < 0.05; GFP-DP71L+Vehicle vs. GFP-DP71L+Poly(I:C): t = 1.15, P > 0.99; GFP+Poly(I:C) vs. GFP- DP71L+Poly(I:C): t = 6.13, P < 0.001], PERK ( E, n = 3-6 per group, F 3,15 = 1.90, GFP+Vehicle vs. GFP+Tg: t = 5.52, P < 0.0001; GFP-DP71L+Vehicle vs. GFP-DP71L+Tg: t = 2.90, P = 0.06; GFP+Tg vs. GFP-DP71L+Tg: t = 5.99, P < 0.001) and GCN2 ( F, n = 3 per group, F 3,8 = 0.67; GFP+Vehicle vs. GFP+Ner: t = 6.29, P < 0.01; GFP-DP71L+Vehicle vs. GFP-DP71L+Ner: t = 2.20, P < 0.35; GFP+Ner vs. GFP-DP71L+Ner: t = 9.27, P < 0.001). Tg = thapsigargin, Oligo = oliogomycin, Ner = neratinib, Poly:IC = polyinosinic-polycytidylic acid: a synthetic double strand RNA. Data are mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: Antibodies used in this study: 119A11 eIF2-P (Cell Signaling Technology, Danvers, MA) or E90 ab32157 eIF2-P (Abcam, Cambridge, UK), D7D3 XP eIF2-T (Cell Signaling Technology, Dnvers, MA), M0802 GFP (Abiocode, Agoura Hills, CA), or A11192 GFP (Thermo Fisher Scientific, Waltham, MA), MABE343 puromycin (MilliporeSigma, Burlington, MA), sc7482 clone E9 PP1α (Santa Cruz Biotechnology, Santa Cruz, CA), PA5-31177 eIF2α (Thermo Fisher Scientific, Waltham, MA), A301-742A eIF2α (Bethyl Laboratories, Montgomery, TX), A300-721A eIF2α (Bethyl Laboratories, Montgomery, TX), 10449-1-AP PPP1R15A (Proteintech Group, Rosemont, IL), 14634-1- AP PPP1R15B (Proteintech Group, Rosemont, IL), 60004-1-IG GAPDH (Proteintech Group, Rosemont, IL).

Techniques: Binding Assay, Expressing, Western Blot, Activation Assay

Journal: bioRxiv

Article Title: Harnessing the Evolution of Proteostasis Networks to Reverse Cognitive Dysfunction

doi: 10.1101/2025.02.28.640897

Figure Lengend Snippet: Molecular dissection of DP71L function. (A) Schematic comparison of DP71L and ΔPPP1R15B, highlighting their PP1- and eIF2-binding motifs. (B) Representative Western blot of eIF2-P levels following expression of either GFP-DP71L or GFP- ΔPPP1R15B. (C) Immunoprecipitation of GFP-DP71L or GFP-ΔPPP1R15B using a GFP antibody, followed by Western blot against endogenous PP1 or eIF2. (D) Schematic of the different GFP-DP71L/GFP-ΔPPP1R15B chimeras. (E) Immunoprecipitation of the different chimeras with a GFP antibody, followed by Western blotting for endogenous PP1. (F) Schematic of various GFP-DP71L and GFP-ΔPPP1R15B chimeras. (G) Immunoprecipitation of the different chimeras using a GFP antibody, followed by Western blotting for endogenous PP1.

Article Snippet: Antibodies used in this study: 119A11 eIF2-P (Cell Signaling Technology, Danvers, MA) or E90 ab32157 eIF2-P (Abcam, Cambridge, UK), D7D3 XP eIF2-T (Cell Signaling Technology, Dnvers, MA), M0802 GFP (Abiocode, Agoura Hills, CA), or A11192 GFP (Thermo Fisher Scientific, Waltham, MA), MABE343 puromycin (MilliporeSigma, Burlington, MA), sc7482 clone E9 PP1α (Santa Cruz Biotechnology, Santa Cruz, CA), PA5-31177 eIF2α (Thermo Fisher Scientific, Waltham, MA), A301-742A eIF2α (Bethyl Laboratories, Montgomery, TX), A300-721A eIF2α (Bethyl Laboratories, Montgomery, TX), 10449-1-AP PPP1R15A (Proteintech Group, Rosemont, IL), 14634-1- AP PPP1R15B (Proteintech Group, Rosemont, IL), 60004-1-IG GAPDH (Proteintech Group, Rosemont, IL).

Techniques: Dissection, Comparison, Binding Assay, Western Blot, Expressing, Immunoprecipitation

Journal: bioRxiv

Article Title: Harnessing the Evolution of Proteostasis Networks to Reverse Cognitive Dysfunction

doi: 10.1101/2025.02.28.640897

Figure Lengend Snippet: Structural characterization of DP71L function. (A) A 3.1 Å electron density map of the PP1•DP71L•eIF2α•G-actin complex (left), and the same map rotated 90°. DP71L is colored blue, PP1 is orange, eIF2α is yellow, and G-actin is copper. (B) Zoomed view of the linker region of DP71L and PPP1R15B, which was modeled into the cryo-EM map (green). (C) Structural modeling a DP71L mutant (4X) predicts an increase the size of the bulge and disruption of the hydrogen bonding, likely leading to decreased binding affinity for PP1. (D) Immunoprecipitation of WT GFP-DP71L and 4X GFP-DP71L with an GFP antibody, followed by Western blotting for endogenous PP1. (E) Structural modeling a mutant ΔPPP1R15B (3X) predicts a decrease in the size of the bulge and the formation of a new hydrogen bond, potentially enhancing binding to PP1. (F) Immunoprecipitation of WT ΔPPP1R15B and 3X ΔPPP1R15B with an GFP antibody, followed by Western blotting for endogenous PP1.

Article Snippet: Antibodies used in this study: 119A11 eIF2-P (Cell Signaling Technology, Danvers, MA) or E90 ab32157 eIF2-P (Abcam, Cambridge, UK), D7D3 XP eIF2-T (Cell Signaling Technology, Dnvers, MA), M0802 GFP (Abiocode, Agoura Hills, CA), or A11192 GFP (Thermo Fisher Scientific, Waltham, MA), MABE343 puromycin (MilliporeSigma, Burlington, MA), sc7482 clone E9 PP1α (Santa Cruz Biotechnology, Santa Cruz, CA), PA5-31177 eIF2α (Thermo Fisher Scientific, Waltham, MA), A301-742A eIF2α (Bethyl Laboratories, Montgomery, TX), A300-721A eIF2α (Bethyl Laboratories, Montgomery, TX), 10449-1-AP PPP1R15A (Proteintech Group, Rosemont, IL), 14634-1- AP PPP1R15B (Proteintech Group, Rosemont, IL), 60004-1-IG GAPDH (Proteintech Group, Rosemont, IL).

Techniques: Cryo-EM Sample Prep, Mutagenesis, Disruption, Binding Assay, Immunoprecipitation, Western Blot