Journal: RNA

Article Title: Variola virus E3L Zα domain, but not its Z-DNA binding activity, is required for PKR inhibition

doi: 10.1261/rna.042341.113

Figure Lengend Snippet: Genetic evidence that the Z-DNA binding domain of E3L is essential for PKR inhibition in yeast. ( A ) Domain architecture of variola E3L and human PKR. (ZBD) Z-DNA binding domain; (dsRBD) dsRNA-binding domain. ( B ) Transformants of yeast strains J110 (−PKR) and H2544 (+PKR) bearing empty vector pEMBLyex4 or plasmids that express the indicated E3L protein under the control of a GAL–CYC1 promoter were grown to saturation, and 5 µL of serial dilutions (of OD 600 = 1.0, 0.1, 0.01, and 0.001) was spotted on SCGal (10% galactose) medium and incubated for 3 d at 30°C. ( C ) Immunoblot analysis of E3L and PKR. WCEs were prepared from the yeast transformants described in B , and 5 µg of total protein extracts was subjected to SDS-PAGE followed by immunoblot analysis using monoclonal antibodies directed against PKR and the HA-epitope to detect N-terminally HA-tagged E3L or polyclonal antisera against yeast eIF2α.

Article Snippet: Five micrograms of WCEs was resolved by SDS-PAGE and subjected to immunoblot analysis using rabbit phosphospecific antibodies directed against phosphorylated Ser51 of eIF2α (BioSource International), rabbit polyclonal antiserum that recognizes yeast eIF2α irrespective of Ser51 phosphorylation, mouse monoclonal anti-HA (Roche), or mouse monoclonal anti-PKR antibodies (R&D Systems).

Techniques: Binding Assay, Inhibition, Plasmid Preparation, Control, Incubation, Western Blot, SDS Page, Bioprocessing

Journal: RNA

Article Title: Variola virus E3L Zα domain, but not its Z-DNA binding activity, is required for PKR inhibition

doi: 10.1261/rna.042341.113

Figure Lengend Snippet: E3L ZBD is required for inhibition of PKR in vitro. ( A ) E3L inhibition of eIF2α phosphorylation; 2.5 nM purified Flag-His 6 -PKR was pre-incubated with 2.5 or 25 nM purified GST (lanes 2 , 3 ), GST-E3L (lanes 4 , 5 ), or GST-E3L-Δ80 (lanes 6 , 7 ) for 10 min at room temperature, then kinase reactions were initiated by addition of 0.2 µM His 6 -tagged eIF2α 1–200 and 0.2 mM ATP. Reactions were resolved by SDS-PAGE and subjected to immunoblot analysis using phosphospecific antibodies against Ser51 in eIF2α ( top panel, eIF2α-P). The membranes were then sequentially stripped and probed using polyclonal antiserum against yeast eIF2α ( second panel, eIF2α), polyclonal anti-GST antiserum ( third panel), and monoclonal anti-PKR antibody ( bottom panel). ( B ) GST-E3L-Δ80 mutation does not impair dsRNA binding in vitro. Purified GST, GST-E3L, and GST-E3L-Δ80 were incubated with poly(I:C)-agarose for 1 h at 4°C. Bound proteins were collected by centrifugation and eluted by boiling in SDS sample buffer. Proteins were resolved by SDS-PAGE and subjected to immunoblot analysis using polyclonal anti-GST antiserum. (T) 5% of total protein used in the assay; (U) unbound (20%); (B) protein bound (20%) to the poly(I:C) agarose.

Article Snippet: Five micrograms of WCEs was resolved by SDS-PAGE and subjected to immunoblot analysis using rabbit phosphospecific antibodies directed against phosphorylated Ser51 of eIF2α (BioSource International), rabbit polyclonal antiserum that recognizes yeast eIF2α irrespective of Ser51 phosphorylation, mouse monoclonal anti-HA (Roche), or mouse monoclonal anti-PKR antibodies (R&D Systems).

Techniques: Inhibition, In Vitro, Phospho-proteomics, Purification, Incubation, SDS Page, Western Blot, Mutagenesis, Binding Assay, Centrifugation

Journal: RNA

Article Title: Variola virus E3L Zα domain, but not its Z-DNA binding activity, is required for PKR inhibition

doi: 10.1261/rna.042341.113

Figure Lengend Snippet: Abundance of dsRNA alters the requirement of the E3L ZBD for PKR inhibition in mammalian cells. ( A ) Human HeLa PKR kd cells were cotransfected with expression vectors for luciferase, an empty vector or knockdown-resistant human PKR (100 ng), and C-terminally HA-tagged variola E3L-HA or E3L-Δ80-HA (400 ng). After 40 h, cells were harvested, lysed, and extracts were assayed for luciferase activity. Luciferase activity was normalized to the transfections containing PKR but lacking E3L. Error bars indicate the standard deviation for three independent transfections. ( B ) Analysis of luciferase activity following cotransfection of PKR kd cells with poly(I:C) (500 ng) and the expression vectors for luciferase, vector or PKR (100 ng), and E3L-HA or E3L-Δ80-HA (400 ng) as described in panel A . ( C ) Immunoblot analyses of WCEs from cells transfected as described in panels A and B with or without poly(I:C) and the indicated expression vectors for PKR, E3L, or E3L-Δ80. Blots were probed with antibodies specific for phosphorylated Ser51 in eIF2α ( top panel), polyclonal antiserum against human eIF2α ( second panel), monoclonal human PKR antibody ( third panel), and monoclonal anti-HA antibody to detect HA-tagged E3L ( fourth panel). Note that E3L-HA resolves as two species on SDS-PAGE (see text). (*) E3L-Δ80 comigrates with a nonspecific protein that cross-reacts with the anti-HA antibody. ( Bottom panel) Relative level of eIF2α-P to total eIF2α was determined for three independent experiments using quantitative densitometry and Image J software (NIH) and normalized to the vector transformant.

Article Snippet: Five micrograms of WCEs was resolved by SDS-PAGE and subjected to immunoblot analysis using rabbit phosphospecific antibodies directed against phosphorylated Ser51 of eIF2α (BioSource International), rabbit polyclonal antiserum that recognizes yeast eIF2α irrespective of Ser51 phosphorylation, mouse monoclonal anti-HA (Roche), or mouse monoclonal anti-PKR antibodies (R&D Systems).

Techniques: Inhibition, Expressing, Luciferase, Plasmid Preparation, Knockdown, Activity Assay, Transfection, Standard Deviation, Cotransfection, Western Blot, SDS Page, Software

Journal: RNA

Article Title: Variola virus E3L Zα domain, but not its Z-DNA binding activity, is required for PKR inhibition

doi: 10.1261/rna.042341.113

Figure Lengend Snippet: E3L inhibition of PKR in yeast is independent of Z-DNA binding activity. ( A ) Transformants of yeast strains J110 (−PKR) and H2544 (+PKR) bearing empty vector pEMBLyex4 or plasmids expressing the indicated N-terminally HA-tagged E3L derivatives under the control of the GAL–CYC1 promoter were grown to saturation, and 5 µL of serial dilutions (of OD 600 = 1.0, 0.1, 0.01, and 0.001) was spotted on SCGal (10% galactose) medium and incubated for 3 d at 30°C. ( B ) Transformants of H2544 (+PKR) from panel A were grown in SD medium at 30°C until OD ∼ 0.6, then shifted to SCGal medium for 2 h. WCEs were fractionated by SDS-PAGE and subjected to immunoblot analysis using monoclonal antibodies against the HA epitope to detect HA-tagged E3L ( upper panel) and polyclonal antisera against yeast eIF2α ( lower panel).

Article Snippet: Five micrograms of WCEs was resolved by SDS-PAGE and subjected to immunoblot analysis using rabbit phosphospecific antibodies directed against phosphorylated Ser51 of eIF2α (BioSource International), rabbit polyclonal antiserum that recognizes yeast eIF2α irrespective of Ser51 phosphorylation, mouse monoclonal anti-HA (Roche), or mouse monoclonal anti-PKR antibodies (R&D Systems).

Techniques: Inhibition, Binding Assay, Activity Assay, Plasmid Preparation, Expressing, Control, Incubation, SDS Page, Western Blot, Bioprocessing

Journal: RNA

Article Title: Variola virus E3L Zα domain, but not its Z-DNA binding activity, is required for PKR inhibition

doi: 10.1261/rna.042341.113

Figure Lengend Snippet: E3L inhibition of PKR in vitro and in mammalian cells treated with dsRNA is independent of Z-DNA binding activity. ( A ) Electrophoretic mobility shift assay of GST-E3L binding to the Z-DNA substrate [ 32 P]-labeled d(G:5 Br C) 20 . Indicated concentrations of GST-E3L were incubated with labeled d(G:5 Br C) 20 in the presence of 30 nM or 300 nM unlabeled Z-DNA (lanes 6 , 7 ) or B-DNA (lanes 8 , 9 ). Positions of the GST-E3L–Z-DNA complex and free probe are indicated. ( B ) Z-DNA binding activity of E3L mutants. GST or the indicated GST-E3L derivative (5 µM) was incubated with the Z-DNA substrate and analyzed as described in A . ( C ) Z-DNA binding mutations do not impair E3L inhibition of PKR in vitro. The indicated GST or GST-E3L derivatives were incubated with purified Flag-His 6 -PKR and then mixed with His 6 -eIF2α 1–200 and ATP. Reactions were resolved by SDS-PAGE and subjected to immunoblot analysis using antibodies specific for the Ser51-phosphorylated form of eIF2α ( top panel) or using polyclonal antiserum against total yeast eIF2α ( middle panel) or GST ( bottom panel). ( D ) Human HeLa PKR kd cells were cotransfected with poly(I:C) (500 ng) and expression vectors for luciferase, knockdown-resistant human PKR (100 ng), and the indicated E3L-HA derivative (400 ng). After 40 h, cells were harvested, lysed, and extracts were assayed for luciferase activity. Luciferase activity was normalized to the transfections containing PKR but lacking E3L. Error bars indicate the standard deviation for three independent transfections. ( E ) Immunoblot analyses of WCEs from cells transfected as described in panel D . Blots were probed with antibodies specific for phosphorylated Ser51 in eIF2α ( top panel), polyclonal antiserum against human eIF2α ( second panel), monoclonal human PKR antibody ( third panel), and monoclonal anti-HA antibody to detect HA-tagged E3L ( bottom panel). The relative level of eIF2α-P to total eIF2α (2α-P/2α) was determined for three independent experiments using quantitative densitometry and Image J software (NIH) and normalized to the vector transformant.

Article Snippet: Five micrograms of WCEs was resolved by SDS-PAGE and subjected to immunoblot analysis using rabbit phosphospecific antibodies directed against phosphorylated Ser51 of eIF2α (BioSource International), rabbit polyclonal antiserum that recognizes yeast eIF2α irrespective of Ser51 phosphorylation, mouse monoclonal anti-HA (Roche), or mouse monoclonal anti-PKR antibodies (R&D Systems).

Techniques: Inhibition, In Vitro, Binding Assay, Activity Assay, Electrophoretic Mobility Shift Assay, Labeling, Incubation, Purification, SDS Page, Western Blot, Expressing, Luciferase, Knockdown, Transfection, Standard Deviation, Software, Plasmid Preparation

Journal: RNA

Article Title: Variola virus E3L Zα domain, but not its Z-DNA binding activity, is required for PKR inhibition

doi: 10.1261/rna.042341.113

Figure Lengend Snippet: Identification of point mutations in the ZBD of E3L that impair PKR inhibition, but not Z-DNA binding. ( A ) Growth phenotypes of E3L mutants identified through random mutagenesis. Transformants of yeast strains J110 (−PKR) or H2544 (+PKR) bearing empty vector pEMBLyex4 or plasmids that express the indicated HA-E3L derivatives under the control of a GAL–CYC1 promoter were grown to saturation, and 5 µL of serial dilutions (of OD 600 = 1.0, 0.1, 0.01, and 0.001) was spotted on SCGal (10% galactose) medium and incubated for 3 d at 30°C. ( B ) Immunoblot analysis of WCEs from yeast strains in panel A . Transformants were grown in SD medium at 30°C to OD 600 ∼ 0.6, and then shifted to SCGal medium for 2 h. WCEs (5 µg) were fractionated by SDS-PAGE and subjected to immunoblot analysis using monoclonal antibodies against the HA epitope to detect HA-E3L ( upper panel) and polyclonal antisera against yeast eIF2α as loading control ( lower panel). ( C ) dsRNA binding. Purified GST or the indicated GST-E3L derivative was incubated with poly(I:C)-agarose for 1 h at 4°C. Bound proteins were collected by centrifugation and eluted by boiling in SDS sample buffer. Proteins were resolved by SDS-PAGE and subjected to immunoblot analysis using polyclonal anti-GST antiserum. (T) 5% of total protein used in the assay; (U) unbound (20%); (B) protein bound (20%) to the poly(I:C) agarose. ( D ) Z-DNA binding activity of E3L ZBD derivatives. The indicated GST-E3L-ZBD (residues 1–80) derivatives (5 µM) were incubated with the Z-DNA substrate [ 32 P]-labeled d(C:5 Br G) 20 and assayed for Z-DNA binding ability by electrophoretic mobility shift assay.

Article Snippet: Five micrograms of WCEs was resolved by SDS-PAGE and subjected to immunoblot analysis using rabbit phosphospecific antibodies directed against phosphorylated Ser51 of eIF2α (BioSource International), rabbit polyclonal antiserum that recognizes yeast eIF2α irrespective of Ser51 phosphorylation, mouse monoclonal anti-HA (Roche), or mouse monoclonal anti-PKR antibodies (R&D Systems).

Techniques: Inhibition, Binding Assay, Mutagenesis, Plasmid Preparation, Control, Incubation, Western Blot, SDS Page, Bioprocessing, Purification, Centrifugation, Activity Assay, Labeling, Electrophoretic Mobility Shift Assay

Journal: Translational Psychiatry

Article Title: Carboxypeptidase E conditional knockout mice exhibit learning and memory deficits and neurodegeneration

doi: 10.1038/s41398-023-02429-y

Figure Lengend Snippet: a – e Western blot analysis of p -TrkB, BDNF, p -mTOR, mTOR, p -AKT, and AKT levels in the hippocampus. f Immunofluorescence of CPE, MAP2, DCX, and GFAP; and the relative fluorescent intensities of g CPE in Sub, h MAP2 in Sub, i MAP2 in hilius, j DCX in DG, k GFAP in DG of WT, CPE flox/− , and CPE flox/flox mice at 100× and 400× (square in the panel). n = 6; * P < 0.05 and ** P < 0.01 compared with WT; values are mean ± SEM.

Article Snippet: After blocking with 5% nonfat milk, the membrane was blotted with antibodies against mouse anti-β-actin antibody (1:1000, CST, 4967S), mouse anti-CPE antibody (1:1000; BD bioscience, 610758), mouse anti-BDNF antibody (1:600, Abcam, UK, ab108319), rabbit anti-p-TrkB antibody (1:1000; Boster, Wuhan, China, BM4437), rabbit anti-AKT (1:2000, Cell Signaling Technology, 4691S), and anti-p-AKT (1:2000, Cell Signaling Technology, 23430S) overnight at 4 °C.

Techniques: Western Blot, Immunofluorescence