Journal: Science Advances

Article Title: The making of a nucleic acid sensor at the dawn of jawed vertebrate evolution

doi: 10.1126/sciadv.ado7464

Figure Lengend Snippet: ( A ) Model for PKR-mediated antiviral innate immunity. Upon recognition of dsRNA produced by virus infection, PKR dimerizes and autophosphorylates, leading to the full catalytic activation of PKR. Activated PKR then phosphorylates eIF2α, converting eIF2α into an inhibitor of its guanine nucleotide exchange factor eIF2B and thereby down-regulating translation. ( B ) The domain architecture of hsPKR (accession: NP_002750.1). hsPKR consists of two tandem dsRBDs and a kinase domain. The numbers indicate the start or termination sites for different domains. ( C ) Large-scale phylogenetic analyses of PKR homologs based on the kinase domain sequences. Major kinase clades are marked in different colors. For more detailed information, see also fig. S1. Support values are shown near the selected nodes. ( D ) Phylogenetic analysis of EIF2AK proteins based on the kinase domain sequences (accession: cl21453) with Wee1-like kinases as the outgroup. The PKR protein group was highlighted in red. Domain architectures of representative proteins are shown. For proteins with more than one kinase domains, kinase domains were partitioned and analyzed independently, and kinase domains analyzed are labeled with solid red rectangles. The distribution for different EIF2AK groups in cellular organisms is shown, and filled and empty circles indicate the presence and absence of corresponding EIF2AK group proteins, respectively. Support values are shown near the selected nodes. ( E ) Phylogenetic relationships of proteins closely related to PKR proteins based on the kinase domain sequences (accession: cl21453). For each protein, domain architecture is shown. Support values are shown near the selected nodes.

Article Snippet: After blocking with either 5% nonfat dry skim milk or 5% bovine serum albumin for 2 hours at room temperature, the membrane was incubated overnight at 4°C with different antibodies diluted in TBS-T [20 mM tris-HCl (pH 7.6), 150 mM NaCl, and 0.1% Tween 20] in turn [1:5000 for mouse anti-HA (Abclonal no. AE008), 1:5000 for mouse anti-GST (Abclonal no. AE001), 1:5000 for rabbit phospho-eIF2α-S51 (Abclonal no. AP0342), or 1:10,000 for β-actin (Proteintech no. 60008-1-lg)].

Techniques: Produced, Virus, Infection, Activation Assay, Labeling

Journal: Science Advances

Article Title: The making of a nucleic acid sensor at the dawn of jawed vertebrate evolution

doi: 10.1126/sciadv.ado7464

Figure Lengend Snippet: Functional analyses of PKR genes from six representative jawed vertebrates, namely, hsPKR of H. sapiens , pbPKR of P. bivittatus , xtPKR of X. tropicalis , paPKR of P. annectens , drPKR of D. rerio , and rtPKR of R. typus , were performed in the yeast system. ( A ) Transformation of wild-type yeast strains with plasmids expressing PKR genes or PKR genes with kinase-dead mutations under the control of a galactose-inducible promoter. After a series of 10-fold dilutions, the transformants were spotted on the surface of solid medium containing glucose or galactose and incubated at 30°C for 48 or 72 hours, respectively. ( B ) Whole-cell extracts from transformants were separated by 10% SDS-PAGE gel and detected by Western blotting with antibodies against HA tag, β-actin, and phosphospecific antibodies against Ser 51 in eIF2α (eIF2α-P), respectively. ( C ) Transformation of eIF2α S51A strains with plasmids expressing PKR genes under the control of a galactose-inducible promoter. After a series of 10-fold dilutions, the transformants were spotted on the surface of solid medium containing galactose and incubated at 30°C for 72 hours. ( D ) Whole-cell extracts from transformants were separated by 10% SDS-PAGE gel and detected by Western blotting with antibodies against HA tag and β-actin, as well as phosphospecific antibodies against Ser 51 in eIF2α (eIF2α-P), respectively.

Article Snippet: After blocking with either 5% nonfat dry skim milk or 5% bovine serum albumin for 2 hours at room temperature, the membrane was incubated overnight at 4°C with different antibodies diluted in TBS-T [20 mM tris-HCl (pH 7.6), 150 mM NaCl, and 0.1% Tween 20] in turn [1:5000 for mouse anti-HA (Abclonal no. AE008), 1:5000 for mouse anti-GST (Abclonal no. AE001), 1:5000 for rabbit phospho-eIF2α-S51 (Abclonal no. AP0342), or 1:10,000 for β-actin (Proteintech no. 60008-1-lg)].

Techniques: Functional Assay, Transformation Assay, Expressing, Control, Incubation, SDS Page, Western Blot

Journal: Science Advances

Article Title: The making of a nucleic acid sensor at the dawn of jawed vertebrate evolution

doi: 10.1126/sciadv.ado7464

Figure Lengend Snippet: Functional analyses of scPKR - sis gene were performed in the yeast system. ( A ) Transformation of wild-type yeast strains with plasmids expressing GST-scPKR-sis and GST-scPKR-sis-KD fusion proteins or GST-scPKR-sis and GST-scPKR-sis-KD fusion proteins with kinase-dead mutations under the control of a galactose-inducible promoter. Yeast transformants were subjected to 10-fold serial dilution and tested for growth on solid medium containing glucose or galactose. ( B ) Whole-cell extracts from transformants were separated by 10% SDS-PAGE gel and detected by Western blotting with antibodies against HA tag, GST tag, and β-actin, as well as phosphospecific antibodies against Ser 51 in eIF2α (eIF2α-P), respectively. ( C ) Plasmids expressing GST-scPKR-sis and GST-scPKR-sis-KD fusion protein under the control of a galactose-inducible promoter were introduced into yeast strain S288C containing eIF2α S51A . Transformants were subjected to 10-fold serial dilution and tested for growth on solid medium containing glucose or galactose. ( D ) Whole-cell extracts from transformants were separated by 10% SDS-PAGE gel and detected by Western blotting with antibodies against GST tag and β-actin, as well as phosphospecific antibodies against Ser 51 in eIF2α (eIF2α-P), respectively.

Article Snippet: After blocking with either 5% nonfat dry skim milk or 5% bovine serum albumin for 2 hours at room temperature, the membrane was incubated overnight at 4°C with different antibodies diluted in TBS-T [20 mM tris-HCl (pH 7.6), 150 mM NaCl, and 0.1% Tween 20] in turn [1:5000 for mouse anti-HA (Abclonal no. AE008), 1:5000 for mouse anti-GST (Abclonal no. AE001), 1:5000 for rabbit phospho-eIF2α-S51 (Abclonal no. AP0342), or 1:10,000 for β-actin (Proteintech no. 60008-1-lg)].

Techniques: Functional Assay, Transformation Assay, Expressing, Control, Serial Dilution, SDS Page, Western Blot

Journal: Science Advances

Article Title: The making of a nucleic acid sensor at the dawn of jawed vertebrate evolution

doi: 10.1126/sciadv.ado7464

Figure Lengend Snippet: ( A , D , F , and H ) Schematic diagram of chimeric PKR construction. Chimeric PKR proteins, cPKR1 to cPKR4, are derived from hsPKR and scPKR-sis. Sequence fragments from different sources are indicated using different colors. The numbers indicate the corresponding sites in source proteins. ( B , E , G and I ) Functional analysis of hsPKR, scPKR-sis, and chimeric PKR proteins in the yeast system. Transformation of wild-type yeast strains with plasmids expressing hsPKR , scPKR-sis , and chimeric PKR genes under the control of a galactose-inducible promoter. After a series of 10-fold dilutions, the transformants were spotted on the surface of solid medium containing glucose or galactose and incubated at 30°C for 48 or 72 hours, respectively. ( C and J ) Western blot analysis was used to detect the expression of corresponding proteins and the phosphorylation of eIF2α. Whole-cell extracts from transformants were separated by 10% SDS-PAGE gel and detected by Western blotting with antibodies against HA tag and β-actin, as well as phosphospecific antibodies against Ser51 in eIF2α (eIF2α-P), respectively.

Article Snippet: After blocking with either 5% nonfat dry skim milk or 5% bovine serum albumin for 2 hours at room temperature, the membrane was incubated overnight at 4°C with different antibodies diluted in TBS-T [20 mM tris-HCl (pH 7.6), 150 mM NaCl, and 0.1% Tween 20] in turn [1:5000 for mouse anti-HA (Abclonal no. AE008), 1:5000 for mouse anti-GST (Abclonal no. AE001), 1:5000 for rabbit phospho-eIF2α-S51 (Abclonal no. AP0342), or 1:10,000 for β-actin (Proteintech no. 60008-1-lg)].

Techniques: Derivative Assay, Sequencing, Functional Assay, Transformation Assay, Expressing, Control, Incubation, Western Blot, Phospho-proteomics, SDS Page

Journal: Science Advances

Article Title: The making of a nucleic acid sensor at the dawn of jawed vertebrate evolution

doi: 10.1126/sciadv.ado7464

Figure Lengend Snippet: PKR proteins probably originated before the LCA of jawed vertebrates, but after the divergence of jawless and jawed vertebrates. Domain shuffling occurred between a dsRBD-containing protein and a preexisting EIF2AK protein that can phosphorylate eIF2α, giving rise to the PKR progenitor. dsRBD duplication and further modifications occurred in the progenitor, generating the functional PKR. The function of PKR is conserved across jawed vertebrates.

Article Snippet: After blocking with either 5% nonfat dry skim milk or 5% bovine serum albumin for 2 hours at room temperature, the membrane was incubated overnight at 4°C with different antibodies diluted in TBS-T [20 mM tris-HCl (pH 7.6), 150 mM NaCl, and 0.1% Tween 20] in turn [1:5000 for mouse anti-HA (Abclonal no. AE008), 1:5000 for mouse anti-GST (Abclonal no. AE001), 1:5000 for rabbit phospho-eIF2α-S51 (Abclonal no. AP0342), or 1:10,000 for β-actin (Proteintech no. 60008-1-lg)].

Techniques: Functional Assay

Journal: Experimental & molecular medicine

Article Title: Sestrin2 protects against cholestatic liver injury by inhibiting endoplasmic reticulum stress and NLRP3 inflammasome-mediated pyroptosis.

doi: 10.1038/s12276-022-00737-9

Figure Lengend Snippet: Fig. 3 Bile acids upregulate Sestrin2 expression via an ATF4- and C/EBP-β-dependent mechanism. A, B HepG2 cells were treated with 200 µM CDCA or 750 µM CA for the indicated times. Cell lysates were immunoblotted with anti-p-eIF2α, anti-eIF2α, and anti-ATF4 antibodies. C Immunofluorescence staining of PDI (green) in HepG2 cells treated with 200 µM CDCA or 750 µM CA for 9 h (n = 3–4). Nuclei were stained with DAPI (blue). Scale bars, 10 μm. D, E Liver tissues were collected from mice 3 days after sham or BDL surgery (n = 4–5 mice per group) and analyzed by immunoblotting with the indicated antibodies. Band intensities were quantified and normalized to GAPDH or total protein intensities. F Immunohistochemical analysis of BiP in liver tissues from mice 3 days after sham or BDL surgery (n = 4–5 mice per group). The boxed areas are magnified in the bottom panels. Scale bars, 50 μm; 10 μm (insets). G, H HepG2 cells were infected with lentiviruses expressing shRNAs targeting luciferase (sh-Luc) or ATF4 (sh-ATF4) and treated with 200 µM CDCA or 750 µM CA for 9 h. Cell lysates were immunoblotted with anti-Sestrin2 and anti-ATF4 antibodies. I, J HepG2 cells were infected with lentiviral sh-Luc or sh-C/EBP-β and treated with 200 µM CDCA or 750 µM CA for 9 h. Cell lysates were immunoblotted with anti-Sestrin2 and anti-C/EBP-β antibodies. GAPDH or β-actin served as loading controls. Numbers below the immunoblot bands indicate fold changes normalized to the control band intensities. The data are representative of one (D–F) or at least three (A–C, G–J) independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t test).

Article Snippet: Immunoblotting was performed using antibodies against human Sestrin2 (Proteintech Group, USA), mouse Sestrin2 (Dr. Jun Hee Lee, University of Michigan); cleaved caspase-3, phospho-eIF2α, PERK, phospho-p70 S6 kinase, p70 S6 kinase, phospho-S6 ribosomal protein, S6 ribosomal protein, phospho-AMPK, AMPK, TSC2, NLRP3 (Cell Signaling Technology, USA), ATF4, C/EBP-β, eIF2α, caspase-1, ASC (Santa Cruz Biotechnology, USA); GSDMD (Abcam), GAPDH (Aviva Systems Biology, USA), and β-actin (Developmental Studies Hybridoma Bank, University of Iowa).

Techniques: Expressing, Staining, Western Blot, Immunohistochemical staining, Infection, Luciferase, Control

Journal: PLoS ONE

Article Title: Signaling Networks Converge on TORC1-SREBP Activity to Promote Endoplasmic Reticulum Homeostasis

doi: 10.1371/journal.pone.0101164

Figure Lengend Snippet: Antibodies used in the study.

Article Snippet: P-eIF2α (S51) , Enzo Biosc. (Rb) , IF/WB , 1∶400/1∶2000.

Techniques:

Journal: British Journal of Cancer

Article Title: Attenuation of muscle atrophy in a murine model of cachexia by inhibition of the dsRNA-dependent protein kinase

doi: 10.1038/sj.bjc.6603704

Figure Lengend Snippet: Western blots of phospho-PKR ( A ) and -eIF2 α ( B ) in gastrocnemius muscle of mice bearing the MAC16 tumour after 5 days treatment with a PKR inhibitor (1 and 5 mg kg −1 ), as described in the legend to . The blots for total PKR and eIF2 α were used as loading controls. The first lane (CON) used gastrocnemius muscle from an NTB control. Representative blots are shown and the densitometric analysis gives the ratio of the phospho to total forms as an average of three separate blots ( n =9). Differences from NTB control are shown as a: P <0.05 or c: P <0.001, whereas differences from the solvent control are indicated as f: P <0.01.

Article Snippet: Rabbit monoclonal antibodies to phospho-eIF2 α (Ser 51) and to phospho-PKR (Thr 446) were purchased from Insight Biotechnology Ltd (London, UK).

Techniques: Western Blot, Control, Solvent

Journal: British Journal of Cancer

Article Title: Attenuation of muscle atrophy in a murine model of cachexia by inhibition of the dsRNA-dependent protein kinase

doi: 10.1038/sj.bjc.6603704

Figure Lengend Snippet: Flow diagram showing how activation of PKR leads to inhibition of protein synthesis through phosphorylation of eIF2 α ; and increased protein degradation through activation of NF- κ B, which would be attenuated by a PKR inhibitor (PKRI).

Article Snippet: Rabbit monoclonal antibodies to phospho-eIF2 α (Ser 51) and to phospho-PKR (Thr 446) were purchased from Insight Biotechnology Ltd (London, UK).

Techniques: Activation Assay, Inhibition, Phospho-proteomics

Journal:

Article Title: Combination of proteasome inhibitors bortezomib and NPI-0052 trigger in vivo synergistic cytotoxicity in multiple myeloma

doi: 10.1182/blood-2007-08-105601

Figure Lengend Snippet: Effects of NPI-0052 plus bortezomib on Hsp's and proteasomal activities. (A) MM.1S cells were treated with NPI-0052 (1 nM), bortezomib (3 nM), or combined NPI-0052 (1 nM) plus bortezomib (3 nM) for 12 hours and harvested; total proteins were then subjected to immunoblot analysis with anti–phospho-JNK or anti–actin Abs. Blots shown are representative of 3 independent experiments. (B) MM.1S cells were treated with NPI-0052 (1 nM), bortezomib (3 nM), or combined NPI-0052 (1 nM) plus bortezomib (3 nM) for 12 hours and harvested; total proteins were then subjected to immunoblot analysis with anti–phospho-eIF2-α, anti–CHOP/GADD153, or anti–actin Abs. Blots shown are representative of 3 independent experiments. (C) MM.1S cells were treated with NPI-0052 (1 nM), bortezomib (3 nM), or combined NPI-0052 (1 nM) plus bortezomib (3 nM) for 24, 48, and 72 hours and harvested; total proteins were then subjected to immunoblot analysis with anti–Hsp-90, anti–Hsp-27, or anti–Hsp-70 Abs. Lysates from HeLA cells served a positive control in immunobloting with Hsp Abs. Blots shown are representative of 2 independent experiments. (D) MM.1S cells were treated with NPI-0052 (1 nM), bortezomib (3 nM), or combined NPI-0052 (1 nM) plus bortezomib (3 nM) for 24 hours and harvested; nuclear extracts were then analyzed for NF-κB activity by using a p65 enzyme-linked immunosorbent assay (ELISA) kit (Active Motif, Carlsbad, CA). (E) MM.1S cells were treated with indicated concentrations of NPI-0052, bortezomib, or combined NPI-0052 plus bortezomib for 30 minutes and harvested; cytosolic extracts were then analyzed for CT-L, C-L, and T-L proteasomal activities. The data are represented as percentage of inhibition compared with vehicle control. Data are presented as means plus or minus SD. (n = 3; P < .05).

Article Snippet: Western blotting Immunoblot analysis was performed using antibodies to caspase-8, caspase-9, caspase-3 (Cell Signaling, Beverly, MA), PARP, Bcl-2, BIM, Hsp-27, Hsp-70, Hsp-90, CHOP (CEBP homology protein), phospho-eIF2-α, phospho-JNK, actin, or tubulin (BD Bioscience Pharmingen, San Diego, CA).

Techniques: Western Blot, Positive Control, Activity Assay, Enzyme-linked Immunosorbent Assay, Inhibition

Journal: PLoS ONE

Article Title: ER Stress Activates NF-κB by Integrating Functions of Basal IKK Activity, IRE1 and PERK

doi: 10.1371/journal.pone.0045078

Figure Lengend Snippet: (A) 786-0 cells have constitutively active NF-κB. WT MEFs or 786-O cells were induced with DTT for up to 7 hrs. NF-κB activity was measured by EMSA. Also, levels of P-eIF2α or total eIF2α were measured by western blot. MEFs and 786-0 cells show similar patterns of eIF2α phosphorylation indicating an intact UPR in 786-0 cells. (B) 786-0 cells have increased basal IKK activity. IKK activity was measured two ways in WT MEFs or 786-0 cells. IKK activity (i) was measured by incubating IKK complex IP'd from cells with recombinant IκBα and γ 32 P-ATP, whereas IKK activity (ii) shows P-IκBα present in cell extracts measured by western blot. Also, protein levels of P-IκBα, total IκBα, IKKß, and actin were determined by Western Blot. (C) Schematic for following experiments. 786-0 cells were treated with an IKK inhibitor, SC514 for 1 h to reduce IKK activity, then cells were treated without or with DTT for up to 5 hours. (D) SC514 decreases IKK activity. 786-O cells were treated with SC514 for 1 hour, and westerns against P-IκBα, total IκBα, and actin were performed. A reduction in P-IκBα and increase in total IκBα were seen corresponding to a decrease in IKK activity. (E) SC514 does not affect UPR signalling. 786-0 cells were incubated with SC514 for 1 h (‘0’), then DTT was added up to 5 hrs. Westerns against P-eIF2α, total eIF2α, and actin were done. P-eIF2α levels did not change upon addition of SC514, but increased with DTT, indicating SC514 does not alter the normal UPR response. ‘U’ indicates untreated conditions. (F) UPR signalling overrides inhibition by SC514. 786-0 cells were incubated with SC514 for 1 hr (‘0’: lanes 2 and 6). Cells were then incubated either without DTT (lanes 3–4) or with DTT (lanes 7–8) still in the presence of SC514. EMSA was performed to measure NF-κB activity. SC514 resulted in a decrease of NF-κB activity (lanes 2–4), but induction of UPR caused activation of NF-κB even in presence of SC514 (lanes 6–8). ‘U’ indicates untreated conditions. Quantitation with standard error is shown using NF-κB activity in untreated 786-0 cells as 100% and represents at least three independent experiments.

Article Snippet: Samples were analyzed by SDS-PAGE and transferred to nitrocellulose and probed with antibodies against NF-κB p65 subunit (Santa Cruz), p50 (Santa Cruz), IκBα (Santa Cruz), phospho-IκBα (Cell Signaling), eIF2α (Cell Signaling), phospho-eIF2α (Stressgen), IKKα (Santa Cruz), IKKβ (Biosource), IKKγ (Santa Cruz), P-IKK (Cell Signalling), TRAF2, (Santa Cruz), P-IKKβ (Cell Signalling), P-JNK (Santa Cruz), and actin (Sigma).

Techniques: Activity Assay, Western Blot, Phospho-proteomics, Recombinant, Incubation, Inhibition, Activation Assay, Quantitation Assay

Journal: PLoS ONE

Article Title: ER Stress Activates NF-κB by Integrating Functions of Basal IKK Activity, IRE1 and PERK

doi: 10.1371/journal.pone.0045078

Figure Lengend Snippet: Under unstressed conditions, IκBα is being synthesized, binds, and inhibits NF-κB. IRE1, through TRAF2, is maintaining basal IKK activity which is responsible for phosphorylating a subset of IκBα leading to proteosomal degradation and basal NF-κB activity. However, most of the NF-κB is sequestered by IκBα. During ER stress, PERK phosphorylation of eIF2α leads to translation repression which then prevents synthesis of new IκBα and contributes to a decrease in IκBα levels and corresponding increase in free NF-κB levels. Additionally, basal IKK activity is responsible for phosphorylating and degrading the existing IκBα, including IκBα bound to NF-κB, causing a more dramatic decrease in IκBα levels resulting in an even greater amount of free NF-κB. Free NF-κB can then translocate to the nucleus to assist in transcriptional activation of stress response genes. During ER stress in cells with decreased basal IKK, such as ire1 −/− cells, basal IKK is considerably reduced. PERK mediated translation inhibition alone is unable to reduce IκBα levels enough to allow for a significant amount of free NF-κB. Thus, combined inputs from both PERK are IRE1 are required for full activation of NF-κB during ER stress. It should be noted that the possibility remains that additional element(s) beyond both PERK induced translation repression and IRE1 regulation of basal IKK/IκBα stability, may also contribute to overall activation of NF-κB during ER stress.

Article Snippet: Samples were analyzed by SDS-PAGE and transferred to nitrocellulose and probed with antibodies against NF-κB p65 subunit (Santa Cruz), p50 (Santa Cruz), IκBα (Santa Cruz), phospho-IκBα (Cell Signaling), eIF2α (Cell Signaling), phospho-eIF2α (Stressgen), IKKα (Santa Cruz), IKKβ (Biosource), IKKγ (Santa Cruz), P-IKK (Cell Signalling), TRAF2, (Santa Cruz), P-IKKβ (Cell Signalling), P-JNK (Santa Cruz), and actin (Sigma).

Techniques: Synthesized, Activity Assay, Phospho-proteomics, Activation Assay, Inhibition