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    Cell Signaling Technology Inc endoplasmic reticulum kinase
    Experiment design. (A) In Experiment 1, the effect of rtPA on the ICH mouse model was investigated. (B) In Experiment 2, the effect and the possible mechanisms of rtPA in the ICH model using primary cortical neurons in vitro were investigated. (C) In Experiment 3, the mechanism of rtPA’s effect on the ICH model in neurons in vitro was examined using the PI3K pathway inhibitor. (D) In Experiment 4, the protein domain that mediates rtPA’s neuroprotective effect in the ICH model in neurons in vitro was investigated. DMSO: Dimethyl sulfoxide; ER: <t>endoplasmic</t> reticulum; H&E staining: hematoxylin & eosin staining; ICH: intracerebral hemorrhage; rtPA: recombinant tissue plasminogen activator; TUNEL: terminal deoxynucleotidyl transferase dUTP nick-end labeling.
    Endoplasmic Reticulum Kinase, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1190 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/perk+3192s/PERK+Rabbit+mAb/pmc12407559-89-88-94
    Average 96 stars, based on 1190 article reviews
    endoplasmic reticulum kinase - by Bioz Stars, 2026-09
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    1) Product Images from "Recombinant tissue plasminogen activator protects neurons after intracerebral hemorrhage through activating the PI3K/AKT/mTOR pathway"

    Article Title: Recombinant tissue plasminogen activator protects neurons after intracerebral hemorrhage through activating the PI3K/AKT/mTOR pathway

    Journal: Neural Regeneration Research

    doi: 10.4103/NRR.NRR-D-23-01953

    Experiment design. (A) In Experiment 1, the effect of rtPA on the ICH mouse model was investigated. (B) In Experiment 2, the effect and the possible mechanisms of rtPA in the ICH model using primary cortical neurons in vitro were investigated. (C) In Experiment 3, the mechanism of rtPA’s effect on the ICH model in neurons in vitro was examined using the PI3K pathway inhibitor. (D) In Experiment 4, the protein domain that mediates rtPA’s neuroprotective effect in the ICH model in neurons in vitro was investigated. DMSO: Dimethyl sulfoxide; ER: endoplasmic reticulum; H&E staining: hematoxylin & eosin staining; ICH: intracerebral hemorrhage; rtPA: recombinant tissue plasminogen activator; TUNEL: terminal deoxynucleotidyl transferase dUTP nick-end labeling.
    Figure Legend Snippet: Experiment design. (A) In Experiment 1, the effect of rtPA on the ICH mouse model was investigated. (B) In Experiment 2, the effect and the possible mechanisms of rtPA in the ICH model using primary cortical neurons in vitro were investigated. (C) In Experiment 3, the mechanism of rtPA’s effect on the ICH model in neurons in vitro was examined using the PI3K pathway inhibitor. (D) In Experiment 4, the protein domain that mediates rtPA’s neuroprotective effect in the ICH model in neurons in vitro was investigated. DMSO: Dimethyl sulfoxide; ER: endoplasmic reticulum; H&E staining: hematoxylin & eosin staining; ICH: intracerebral hemorrhage; rtPA: recombinant tissue plasminogen activator; TUNEL: terminal deoxynucleotidyl transferase dUTP nick-end labeling.

    Techniques Used: In Vitro, Staining, Recombinant, TUNEL Assay

    rtPA attenuates neurological behavior impairment and apoptosis after ICH. (A–C) Left forelimb placement experiment, corner turn experiment, and modified Garcia score testing were conducted at 1 hour before surgery and 6, 24, and 72 hours after surgery ( n = 14 per group). (D) H&E staining (top) and Nissl staining (bottom) of peri-hematoma tissue at 72 hours after ICH and rtPA treatments ( n = 3 per group). Scale bars: 100 µm. (E) Representative picture of TUNEL staining of peri-hematoma tissue conducted at 72 hours after ICH and rtPA treatments ( n = 3–6 per group). Scale bars: 100 µm. (F) The proportion of TUNEL-positive cells to all nucleated cells surrounding the hematoma ( n = 3–6 per group). (G–J) Analysis of apoptosis-associated proteins at 24 and 72 hours after treatment ( n = 3). Data are represented as mean ± SEM. * P < 0.05, **** P < 0.0001, vs. sham group; &P < 0.05, && P < 0.01, &&&& P < 0.0001, vs . ICH group; # P < 0.05, ## P < 0.01, ### P < 0.001, vs . ICH + vehicle group (two-way analysis of variance followed by Bonferroni post hoc test (A–C) or one-way analysis of variance followed by Tukey’s post hoc test (F, I, J). bax: Apoptosis regulator bax; bcl2: apoptosis regulator bcl2; DAPI: 4′,6-diamidino-2-phenylindole; ER: endoplasmic reticulum; H&E staining: hematoxylin & eosin staining; ICH: intracerebral hemorrhage; rtPA: recombinant tissue plasminogen activator; TUNEL: terminal deoxynucleotidyl transferase dUTP nick-end labeling.
    Figure Legend Snippet: rtPA attenuates neurological behavior impairment and apoptosis after ICH. (A–C) Left forelimb placement experiment, corner turn experiment, and modified Garcia score testing were conducted at 1 hour before surgery and 6, 24, and 72 hours after surgery ( n = 14 per group). (D) H&E staining (top) and Nissl staining (bottom) of peri-hematoma tissue at 72 hours after ICH and rtPA treatments ( n = 3 per group). Scale bars: 100 µm. (E) Representative picture of TUNEL staining of peri-hematoma tissue conducted at 72 hours after ICH and rtPA treatments ( n = 3–6 per group). Scale bars: 100 µm. (F) The proportion of TUNEL-positive cells to all nucleated cells surrounding the hematoma ( n = 3–6 per group). (G–J) Analysis of apoptosis-associated proteins at 24 and 72 hours after treatment ( n = 3). Data are represented as mean ± SEM. * P < 0.05, **** P < 0.0001, vs. sham group; &P < 0.05, && P < 0.01, &&&& P < 0.0001, vs . ICH group; # P < 0.05, ## P < 0.01, ### P < 0.001, vs . ICH + vehicle group (two-way analysis of variance followed by Bonferroni post hoc test (A–C) or one-way analysis of variance followed by Tukey’s post hoc test (F, I, J). bax: Apoptosis regulator bax; bcl2: apoptosis regulator bcl2; DAPI: 4′,6-diamidino-2-phenylindole; ER: endoplasmic reticulum; H&E staining: hematoxylin & eosin staining; ICH: intracerebral hemorrhage; rtPA: recombinant tissue plasminogen activator; TUNEL: terminal deoxynucleotidyl transferase dUTP nick-end labeling.

    Techniques Used: Modification, Staining, TUNEL Assay, Recombinant

    rtPA attenuates neuron apoptosis and autophagy after experimental ICH in vitro . (A–C) The DEGs between control group and hemin group associated with autophagy animals (KEGG: mmu04140), positive regulation of neuron apoptotic process (GO: 0043525), and positive regulation of response to endoplasmic reticulum stress (GO: 1905898) were screened, and the transcriptional levels of DEGs in each group are presented as heatmaps. (D, E) Analysis of apoptosis-associated proteins. (F) Transmission electron microscopy images of neurons after hemin and rtPA treatment. Red asterisk indicates the autophagosome, black arrows indicate the endoplasmic reticulum, and N means nucleus. Scale bars: 1 µm. (G–J) Analysis of autophagy-associated proteins. Data are shown as mean ± SEM ( n = 3–4). * P < 0.05, ** P < 0.01, *** P < 0.001, vs . control group; # P < 0.05, ## P < 0.01, ### P < 0.001, vs. hemin group (one-way analysis of variance followed by Tukey’s post hoc test). bax: Apoptosis regulator bax; bcl2: apoptosis regulator bcl2; beclin1: coiled-coil myosin-like bcl2-interacting protein; DEGs: differential expression genes; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; LC3: microtubule-associated proteins 1A/1B light chain 3B; p62: sequestosome-1/ubiquitin-binding protein p62; rtPA: recombinant tissue plasminogen activator.
    Figure Legend Snippet: rtPA attenuates neuron apoptosis and autophagy after experimental ICH in vitro . (A–C) The DEGs between control group and hemin group associated with autophagy animals (KEGG: mmu04140), positive regulation of neuron apoptotic process (GO: 0043525), and positive regulation of response to endoplasmic reticulum stress (GO: 1905898) were screened, and the transcriptional levels of DEGs in each group are presented as heatmaps. (D, E) Analysis of apoptosis-associated proteins. (F) Transmission electron microscopy images of neurons after hemin and rtPA treatment. Red asterisk indicates the autophagosome, black arrows indicate the endoplasmic reticulum, and N means nucleus. Scale bars: 1 µm. (G–J) Analysis of autophagy-associated proteins. Data are shown as mean ± SEM ( n = 3–4). * P < 0.05, ** P < 0.01, *** P < 0.001, vs . control group; # P < 0.05, ## P < 0.01, ### P < 0.001, vs. hemin group (one-way analysis of variance followed by Tukey’s post hoc test). bax: Apoptosis regulator bax; bcl2: apoptosis regulator bcl2; beclin1: coiled-coil myosin-like bcl2-interacting protein; DEGs: differential expression genes; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; LC3: microtubule-associated proteins 1A/1B light chain 3B; p62: sequestosome-1/ubiquitin-binding protein p62; rtPA: recombinant tissue plasminogen activator.

    Techniques Used: In Vitro, Control, Transmission Assay, Electron Microscopy, Quantitative Proteomics, Ubiquitin Proteomics, Binding Assay, Recombinant

    rtPA ameliorates endoplasmic reticulum stress in the in vitro ICH cell model. (A) Confocal images and three-dimensional reconstruction of endoplasmic reticulum continuity of neurons by ER tracker after hemin and rtPA treatment. Scale bars: 3 µm. (B–F) Quantitative analysis of ER stress–associated proteins of neurons. Data are shown as mean ± SEM ( n = 3 per group). * P < 0.05, ** P < 0.01, vs. control group; # P < 0.05, ## P < 0.01, vs . hemin group (one-way analysis of variance followed by Tukey’s post hoc test). (G) Immunofluorescence staining images of p-PERK (red, labeled by Cy3) in neurons after hemin and rtPA treatment. Scale bars: 50 µm. 3D: Three-dimensional; ATF6: cyclic AMP-dependent transcription factor ATF-6 alpha; DAPI: 4′,6-diamidino-2-phenylindole; eIF2α: eukaryotic translation initiation factor 2 subunit alpha; ER: endoplasmic reticulum; Grp78: endoplasmic reticulum chaperone BiP; PERK: PRKR-like endoplasmic reticulum kinase; rtPA: recombinant tissue plasminogen activator.
    Figure Legend Snippet: rtPA ameliorates endoplasmic reticulum stress in the in vitro ICH cell model. (A) Confocal images and three-dimensional reconstruction of endoplasmic reticulum continuity of neurons by ER tracker after hemin and rtPA treatment. Scale bars: 3 µm. (B–F) Quantitative analysis of ER stress–associated proteins of neurons. Data are shown as mean ± SEM ( n = 3 per group). * P < 0.05, ** P < 0.01, vs. control group; # P < 0.05, ## P < 0.01, vs . hemin group (one-way analysis of variance followed by Tukey’s post hoc test). (G) Immunofluorescence staining images of p-PERK (red, labeled by Cy3) in neurons after hemin and rtPA treatment. Scale bars: 50 µm. 3D: Three-dimensional; ATF6: cyclic AMP-dependent transcription factor ATF-6 alpha; DAPI: 4′,6-diamidino-2-phenylindole; eIF2α: eukaryotic translation initiation factor 2 subunit alpha; ER: endoplasmic reticulum; Grp78: endoplasmic reticulum chaperone BiP; PERK: PRKR-like endoplasmic reticulum kinase; rtPA: recombinant tissue plasminogen activator.

    Techniques Used: In Vitro, Control, Immunofluorescence, Staining, Labeling, Recombinant

    The PI3K inhibitor LY294002 reverses the anti-ER stress effect of rtPA and the EGF domain of rtPA may mediate the PI3K/AKT pathway in the ICH in vitro cell model. (A–C) Analysis of ER stress–associated proteins ( n = 3 per group). (D) Confocal images and three-dimensional reconstruction of endoplasmic reticulum continuity of neurons by ER tracker after rtPA and PI3K inhibitor LY294002 treatment. Scale bars: 3 µm. (E) Immunofluorescence images of p-PERK (red, labeled by Cy3) in neurons after rtPA and PI3K inhibitor LY294002 treatment. Scale bars: 50 µm. (F–H) Analysis of PI3K p85 and p-AKT. Data are represented as mean ± SEM ( n = 3 per group). * P < 0.05, ** P < 0.01, *** P < 0.001, vs . hemin group; & P < 0.05, && P < 0.01, vs . hemin + rtPA group; # P < 0.05, vs . hemin + rtPA + DMSO group (one-way analysis of variance followed by Tukey’s post hoc test). (I) Transmission electron microscopy images of cells after rtPA and rtPA domain inhibitor treatment. Scale bar: 100 µm. 3D: Three-dimensional; AKT: RAC-alpha serine/threonine-protein kinase; ATF6: cyclic AMP-dependent transcription factor ATF-6 alpha; DAPI: 4′,6-diamidino-2-phenylindole; EGF: epidermal growth factor; eIF2α: eukaryotic translation initiation factor 2 subunit alpha; ER: endoplasmic reticulum; LY294002: PI3K inhibitor; mTOR: mammalian target of rapamycin; PERK: PRKR-like endoplasmic reticulum kinase; PI3K: phosphatidylinositol 3-kinase regulatory subunit alpha; rtPA: recombinant tissue plasminogen activator.
    Figure Legend Snippet: The PI3K inhibitor LY294002 reverses the anti-ER stress effect of rtPA and the EGF domain of rtPA may mediate the PI3K/AKT pathway in the ICH in vitro cell model. (A–C) Analysis of ER stress–associated proteins ( n = 3 per group). (D) Confocal images and three-dimensional reconstruction of endoplasmic reticulum continuity of neurons by ER tracker after rtPA and PI3K inhibitor LY294002 treatment. Scale bars: 3 µm. (E) Immunofluorescence images of p-PERK (red, labeled by Cy3) in neurons after rtPA and PI3K inhibitor LY294002 treatment. Scale bars: 50 µm. (F–H) Analysis of PI3K p85 and p-AKT. Data are represented as mean ± SEM ( n = 3 per group). * P < 0.05, ** P < 0.01, *** P < 0.001, vs . hemin group; & P < 0.05, && P < 0.01, vs . hemin + rtPA group; # P < 0.05, vs . hemin + rtPA + DMSO group (one-way analysis of variance followed by Tukey’s post hoc test). (I) Transmission electron microscopy images of cells after rtPA and rtPA domain inhibitor treatment. Scale bar: 100 µm. 3D: Three-dimensional; AKT: RAC-alpha serine/threonine-protein kinase; ATF6: cyclic AMP-dependent transcription factor ATF-6 alpha; DAPI: 4′,6-diamidino-2-phenylindole; EGF: epidermal growth factor; eIF2α: eukaryotic translation initiation factor 2 subunit alpha; ER: endoplasmic reticulum; LY294002: PI3K inhibitor; mTOR: mammalian target of rapamycin; PERK: PRKR-like endoplasmic reticulum kinase; PI3K: phosphatidylinositol 3-kinase regulatory subunit alpha; rtPA: recombinant tissue plasminogen activator.

    Techniques Used: In Vitro, Immunofluorescence, Labeling, Transmission Assay, Electron Microscopy, Recombinant

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    Experiment design. (A) In Experiment 1, the effect of rtPA on the ICH mouse model was investigated. (B) In Experiment 2, the effect and the possible mechanisms of rtPA in the ICH model using primary cortical neurons in vitro were investigated. (C) In Experiment 3, the mechanism of rtPA’s effect on the ICH model in neurons in vitro was examined using the PI3K pathway inhibitor. (D) In Experiment 4, the protein domain that mediates rtPA’s neuroprotective effect in the ICH model in neurons in vitro was investigated. DMSO: Dimethyl sulfoxide; ER: endoplasmic reticulum; H&E staining: hematoxylin & eosin staining; ICH: intracerebral hemorrhage; rtPA: recombinant tissue plasminogen activator; TUNEL: terminal deoxynucleotidyl transferase dUTP nick-end labeling.

    Journal: Neural Regeneration Research

    Article Title: Recombinant tissue plasminogen activator protects neurons after intracerebral hemorrhage through activating the PI3K/AKT/mTOR pathway

    doi: 10.4103/NRR.NRR-D-23-01953

    Figure Lengend Snippet: Experiment design. (A) In Experiment 1, the effect of rtPA on the ICH mouse model was investigated. (B) In Experiment 2, the effect and the possible mechanisms of rtPA in the ICH model using primary cortical neurons in vitro were investigated. (C) In Experiment 3, the mechanism of rtPA’s effect on the ICH model in neurons in vitro was examined using the PI3K pathway inhibitor. (D) In Experiment 4, the protein domain that mediates rtPA’s neuroprotective effect in the ICH model in neurons in vitro was investigated. DMSO: Dimethyl sulfoxide; ER: endoplasmic reticulum; H&E staining: hematoxylin & eosin staining; ICH: intracerebral hemorrhage; rtPA: recombinant tissue plasminogen activator; TUNEL: terminal deoxynucleotidyl transferase dUTP nick-end labeling.

    Article Snippet: The following primary antibodies were used for analysis: bcl2 (rabbit, 1:1000, Proteintech, Cat# 12789-1-AP, RRID: AB_2227948), bax (rabbit, 1:1000, Proteintech, Cat# 50599-2-Ig, RRID: AB_2061561), coiled-coil myosin-like bcl2-interacting protein (beclin1; rabbit, 1:1000, Proteintech, Cat# 11306-1-AP, RRID: AB_2259061), sequestosome-1/ubiquitin-binding protein p62 (SQSTM1/p62; rabbit, 1:1000, Abclonal, Cat# A11250, RRID: AB_2758477), microtubule-associated proteins 1A/1B light chain 3B (LC3; rabbit, 1:1000, Abcam, Cat# ab48394, RRID: AB_881433), endoplasmic reticulum chaperone BiP (Grp78/BIP; mouse, 1:1000, Proteintech, Cat# 66574-1-Ig, RRID: AB_2881934), cyclic AMP-dependent transcription factor ATF-6 alpha (ATF6; rabbit, 1:1000, Proteintech, Cat# 24169-1-AP, RRID: AB_2876891), PRKR-like endoplasmic reticulum kinase (PERK; rabbit, 1:1000, Cell Signaling Technology, Cat# 3192S, RRID: AB_2095847), phospho-PERK (rabbit, 1:1000, Cell Signaling Technology, Cat# 3179S, RRID: AB_2095853), eukaryotic translation initiation factor 2 subunit alpha (eIF2α; rabbit, 1:1000, Cell Signaling Technology, Cat# 9722S, RRID: AB_2230924), phospho-eIF2α (rabbit, 1:1000, Cell Signaling Technology, 9721S, RRID: AB_330951), phosphatidylinositol 3-kinase regulatory subunit alpha (PI3 kinase p85; rabbit, 1:1000, Cell Signaling Technology, Cat# 4257S, RRID: AB_659889), RAC-alpha serine/threonine-protein kinase (AKT; rabbit, 1:1000, Cell Signaling Technology, Cat# 4691S, RRID: AB_915783), phospho-AKT (rabbit, 1:1000, Cell Signaling Technology, Cat# 4060S, RRID: AB_2315049), mammalian target of rapamycin (mTOR; rabbit, 1:1000, Cell Signaling Technology, Cat# 2983S, RRID: AB_2105622), phospho-mTOR (rabbit, 1:1000, Cell Signaling Technology, Cat# 2971S, RRID: AB_330970), and β-actin (mouse, 1:1000, Proteintech, Cat# 66009-1-Ig, RRID: AB_2687938).

    Techniques: In Vitro, Staining, Recombinant, TUNEL Assay

    rtPA attenuates neurological behavior impairment and apoptosis after ICH. (A–C) Left forelimb placement experiment, corner turn experiment, and modified Garcia score testing were conducted at 1 hour before surgery and 6, 24, and 72 hours after surgery ( n = 14 per group). (D) H&E staining (top) and Nissl staining (bottom) of peri-hematoma tissue at 72 hours after ICH and rtPA treatments ( n = 3 per group). Scale bars: 100 µm. (E) Representative picture of TUNEL staining of peri-hematoma tissue conducted at 72 hours after ICH and rtPA treatments ( n = 3–6 per group). Scale bars: 100 µm. (F) The proportion of TUNEL-positive cells to all nucleated cells surrounding the hematoma ( n = 3–6 per group). (G–J) Analysis of apoptosis-associated proteins at 24 and 72 hours after treatment ( n = 3). Data are represented as mean ± SEM. * P < 0.05, **** P < 0.0001, vs. sham group; &P < 0.05, && P < 0.01, &&&& P < 0.0001, vs . ICH group; # P < 0.05, ## P < 0.01, ### P < 0.001, vs . ICH + vehicle group (two-way analysis of variance followed by Bonferroni post hoc test (A–C) or one-way analysis of variance followed by Tukey’s post hoc test (F, I, J). bax: Apoptosis regulator bax; bcl2: apoptosis regulator bcl2; DAPI: 4′,6-diamidino-2-phenylindole; ER: endoplasmic reticulum; H&E staining: hematoxylin & eosin staining; ICH: intracerebral hemorrhage; rtPA: recombinant tissue plasminogen activator; TUNEL: terminal deoxynucleotidyl transferase dUTP nick-end labeling.

    Journal: Neural Regeneration Research

    Article Title: Recombinant tissue plasminogen activator protects neurons after intracerebral hemorrhage through activating the PI3K/AKT/mTOR pathway

    doi: 10.4103/NRR.NRR-D-23-01953

    Figure Lengend Snippet: rtPA attenuates neurological behavior impairment and apoptosis after ICH. (A–C) Left forelimb placement experiment, corner turn experiment, and modified Garcia score testing were conducted at 1 hour before surgery and 6, 24, and 72 hours after surgery ( n = 14 per group). (D) H&E staining (top) and Nissl staining (bottom) of peri-hematoma tissue at 72 hours after ICH and rtPA treatments ( n = 3 per group). Scale bars: 100 µm. (E) Representative picture of TUNEL staining of peri-hematoma tissue conducted at 72 hours after ICH and rtPA treatments ( n = 3–6 per group). Scale bars: 100 µm. (F) The proportion of TUNEL-positive cells to all nucleated cells surrounding the hematoma ( n = 3–6 per group). (G–J) Analysis of apoptosis-associated proteins at 24 and 72 hours after treatment ( n = 3). Data are represented as mean ± SEM. * P < 0.05, **** P < 0.0001, vs. sham group; &P < 0.05, && P < 0.01, &&&& P < 0.0001, vs . ICH group; # P < 0.05, ## P < 0.01, ### P < 0.001, vs . ICH + vehicle group (two-way analysis of variance followed by Bonferroni post hoc test (A–C) or one-way analysis of variance followed by Tukey’s post hoc test (F, I, J). bax: Apoptosis regulator bax; bcl2: apoptosis regulator bcl2; DAPI: 4′,6-diamidino-2-phenylindole; ER: endoplasmic reticulum; H&E staining: hematoxylin & eosin staining; ICH: intracerebral hemorrhage; rtPA: recombinant tissue plasminogen activator; TUNEL: terminal deoxynucleotidyl transferase dUTP nick-end labeling.

    Article Snippet: The following primary antibodies were used for analysis: bcl2 (rabbit, 1:1000, Proteintech, Cat# 12789-1-AP, RRID: AB_2227948), bax (rabbit, 1:1000, Proteintech, Cat# 50599-2-Ig, RRID: AB_2061561), coiled-coil myosin-like bcl2-interacting protein (beclin1; rabbit, 1:1000, Proteintech, Cat# 11306-1-AP, RRID: AB_2259061), sequestosome-1/ubiquitin-binding protein p62 (SQSTM1/p62; rabbit, 1:1000, Abclonal, Cat# A11250, RRID: AB_2758477), microtubule-associated proteins 1A/1B light chain 3B (LC3; rabbit, 1:1000, Abcam, Cat# ab48394, RRID: AB_881433), endoplasmic reticulum chaperone BiP (Grp78/BIP; mouse, 1:1000, Proteintech, Cat# 66574-1-Ig, RRID: AB_2881934), cyclic AMP-dependent transcription factor ATF-6 alpha (ATF6; rabbit, 1:1000, Proteintech, Cat# 24169-1-AP, RRID: AB_2876891), PRKR-like endoplasmic reticulum kinase (PERK; rabbit, 1:1000, Cell Signaling Technology, Cat# 3192S, RRID: AB_2095847), phospho-PERK (rabbit, 1:1000, Cell Signaling Technology, Cat# 3179S, RRID: AB_2095853), eukaryotic translation initiation factor 2 subunit alpha (eIF2α; rabbit, 1:1000, Cell Signaling Technology, Cat# 9722S, RRID: AB_2230924), phospho-eIF2α (rabbit, 1:1000, Cell Signaling Technology, 9721S, RRID: AB_330951), phosphatidylinositol 3-kinase regulatory subunit alpha (PI3 kinase p85; rabbit, 1:1000, Cell Signaling Technology, Cat# 4257S, RRID: AB_659889), RAC-alpha serine/threonine-protein kinase (AKT; rabbit, 1:1000, Cell Signaling Technology, Cat# 4691S, RRID: AB_915783), phospho-AKT (rabbit, 1:1000, Cell Signaling Technology, Cat# 4060S, RRID: AB_2315049), mammalian target of rapamycin (mTOR; rabbit, 1:1000, Cell Signaling Technology, Cat# 2983S, RRID: AB_2105622), phospho-mTOR (rabbit, 1:1000, Cell Signaling Technology, Cat# 2971S, RRID: AB_330970), and β-actin (mouse, 1:1000, Proteintech, Cat# 66009-1-Ig, RRID: AB_2687938).

    Techniques: Modification, Staining, TUNEL Assay, Recombinant

    rtPA attenuates neuron apoptosis and autophagy after experimental ICH in vitro . (A–C) The DEGs between control group and hemin group associated with autophagy animals (KEGG: mmu04140), positive regulation of neuron apoptotic process (GO: 0043525), and positive regulation of response to endoplasmic reticulum stress (GO: 1905898) were screened, and the transcriptional levels of DEGs in each group are presented as heatmaps. (D, E) Analysis of apoptosis-associated proteins. (F) Transmission electron microscopy images of neurons after hemin and rtPA treatment. Red asterisk indicates the autophagosome, black arrows indicate the endoplasmic reticulum, and N means nucleus. Scale bars: 1 µm. (G–J) Analysis of autophagy-associated proteins. Data are shown as mean ± SEM ( n = 3–4). * P < 0.05, ** P < 0.01, *** P < 0.001, vs . control group; # P < 0.05, ## P < 0.01, ### P < 0.001, vs. hemin group (one-way analysis of variance followed by Tukey’s post hoc test). bax: Apoptosis regulator bax; bcl2: apoptosis regulator bcl2; beclin1: coiled-coil myosin-like bcl2-interacting protein; DEGs: differential expression genes; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; LC3: microtubule-associated proteins 1A/1B light chain 3B; p62: sequestosome-1/ubiquitin-binding protein p62; rtPA: recombinant tissue plasminogen activator.

    Journal: Neural Regeneration Research

    Article Title: Recombinant tissue plasminogen activator protects neurons after intracerebral hemorrhage through activating the PI3K/AKT/mTOR pathway

    doi: 10.4103/NRR.NRR-D-23-01953

    Figure Lengend Snippet: rtPA attenuates neuron apoptosis and autophagy after experimental ICH in vitro . (A–C) The DEGs between control group and hemin group associated with autophagy animals (KEGG: mmu04140), positive regulation of neuron apoptotic process (GO: 0043525), and positive regulation of response to endoplasmic reticulum stress (GO: 1905898) were screened, and the transcriptional levels of DEGs in each group are presented as heatmaps. (D, E) Analysis of apoptosis-associated proteins. (F) Transmission electron microscopy images of neurons after hemin and rtPA treatment. Red asterisk indicates the autophagosome, black arrows indicate the endoplasmic reticulum, and N means nucleus. Scale bars: 1 µm. (G–J) Analysis of autophagy-associated proteins. Data are shown as mean ± SEM ( n = 3–4). * P < 0.05, ** P < 0.01, *** P < 0.001, vs . control group; # P < 0.05, ## P < 0.01, ### P < 0.001, vs. hemin group (one-way analysis of variance followed by Tukey’s post hoc test). bax: Apoptosis regulator bax; bcl2: apoptosis regulator bcl2; beclin1: coiled-coil myosin-like bcl2-interacting protein; DEGs: differential expression genes; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; LC3: microtubule-associated proteins 1A/1B light chain 3B; p62: sequestosome-1/ubiquitin-binding protein p62; rtPA: recombinant tissue plasminogen activator.

    Article Snippet: The following primary antibodies were used for analysis: bcl2 (rabbit, 1:1000, Proteintech, Cat# 12789-1-AP, RRID: AB_2227948), bax (rabbit, 1:1000, Proteintech, Cat# 50599-2-Ig, RRID: AB_2061561), coiled-coil myosin-like bcl2-interacting protein (beclin1; rabbit, 1:1000, Proteintech, Cat# 11306-1-AP, RRID: AB_2259061), sequestosome-1/ubiquitin-binding protein p62 (SQSTM1/p62; rabbit, 1:1000, Abclonal, Cat# A11250, RRID: AB_2758477), microtubule-associated proteins 1A/1B light chain 3B (LC3; rabbit, 1:1000, Abcam, Cat# ab48394, RRID: AB_881433), endoplasmic reticulum chaperone BiP (Grp78/BIP; mouse, 1:1000, Proteintech, Cat# 66574-1-Ig, RRID: AB_2881934), cyclic AMP-dependent transcription factor ATF-6 alpha (ATF6; rabbit, 1:1000, Proteintech, Cat# 24169-1-AP, RRID: AB_2876891), PRKR-like endoplasmic reticulum kinase (PERK; rabbit, 1:1000, Cell Signaling Technology, Cat# 3192S, RRID: AB_2095847), phospho-PERK (rabbit, 1:1000, Cell Signaling Technology, Cat# 3179S, RRID: AB_2095853), eukaryotic translation initiation factor 2 subunit alpha (eIF2α; rabbit, 1:1000, Cell Signaling Technology, Cat# 9722S, RRID: AB_2230924), phospho-eIF2α (rabbit, 1:1000, Cell Signaling Technology, 9721S, RRID: AB_330951), phosphatidylinositol 3-kinase regulatory subunit alpha (PI3 kinase p85; rabbit, 1:1000, Cell Signaling Technology, Cat# 4257S, RRID: AB_659889), RAC-alpha serine/threonine-protein kinase (AKT; rabbit, 1:1000, Cell Signaling Technology, Cat# 4691S, RRID: AB_915783), phospho-AKT (rabbit, 1:1000, Cell Signaling Technology, Cat# 4060S, RRID: AB_2315049), mammalian target of rapamycin (mTOR; rabbit, 1:1000, Cell Signaling Technology, Cat# 2983S, RRID: AB_2105622), phospho-mTOR (rabbit, 1:1000, Cell Signaling Technology, Cat# 2971S, RRID: AB_330970), and β-actin (mouse, 1:1000, Proteintech, Cat# 66009-1-Ig, RRID: AB_2687938).

    Techniques: In Vitro, Control, Transmission Assay, Electron Microscopy, Quantitative Proteomics, Ubiquitin Proteomics, Binding Assay, Recombinant

    rtPA ameliorates endoplasmic reticulum stress in the in vitro ICH cell model. (A) Confocal images and three-dimensional reconstruction of endoplasmic reticulum continuity of neurons by ER tracker after hemin and rtPA treatment. Scale bars: 3 µm. (B–F) Quantitative analysis of ER stress–associated proteins of neurons. Data are shown as mean ± SEM ( n = 3 per group). * P < 0.05, ** P < 0.01, vs. control group; # P < 0.05, ## P < 0.01, vs . hemin group (one-way analysis of variance followed by Tukey’s post hoc test). (G) Immunofluorescence staining images of p-PERK (red, labeled by Cy3) in neurons after hemin and rtPA treatment. Scale bars: 50 µm. 3D: Three-dimensional; ATF6: cyclic AMP-dependent transcription factor ATF-6 alpha; DAPI: 4′,6-diamidino-2-phenylindole; eIF2α: eukaryotic translation initiation factor 2 subunit alpha; ER: endoplasmic reticulum; Grp78: endoplasmic reticulum chaperone BiP; PERK: PRKR-like endoplasmic reticulum kinase; rtPA: recombinant tissue plasminogen activator.

    Journal: Neural Regeneration Research

    Article Title: Recombinant tissue plasminogen activator protects neurons after intracerebral hemorrhage through activating the PI3K/AKT/mTOR pathway

    doi: 10.4103/NRR.NRR-D-23-01953

    Figure Lengend Snippet: rtPA ameliorates endoplasmic reticulum stress in the in vitro ICH cell model. (A) Confocal images and three-dimensional reconstruction of endoplasmic reticulum continuity of neurons by ER tracker after hemin and rtPA treatment. Scale bars: 3 µm. (B–F) Quantitative analysis of ER stress–associated proteins of neurons. Data are shown as mean ± SEM ( n = 3 per group). * P < 0.05, ** P < 0.01, vs. control group; # P < 0.05, ## P < 0.01, vs . hemin group (one-way analysis of variance followed by Tukey’s post hoc test). (G) Immunofluorescence staining images of p-PERK (red, labeled by Cy3) in neurons after hemin and rtPA treatment. Scale bars: 50 µm. 3D: Three-dimensional; ATF6: cyclic AMP-dependent transcription factor ATF-6 alpha; DAPI: 4′,6-diamidino-2-phenylindole; eIF2α: eukaryotic translation initiation factor 2 subunit alpha; ER: endoplasmic reticulum; Grp78: endoplasmic reticulum chaperone BiP; PERK: PRKR-like endoplasmic reticulum kinase; rtPA: recombinant tissue plasminogen activator.

    Article Snippet: The following primary antibodies were used for analysis: bcl2 (rabbit, 1:1000, Proteintech, Cat# 12789-1-AP, RRID: AB_2227948), bax (rabbit, 1:1000, Proteintech, Cat# 50599-2-Ig, RRID: AB_2061561), coiled-coil myosin-like bcl2-interacting protein (beclin1; rabbit, 1:1000, Proteintech, Cat# 11306-1-AP, RRID: AB_2259061), sequestosome-1/ubiquitin-binding protein p62 (SQSTM1/p62; rabbit, 1:1000, Abclonal, Cat# A11250, RRID: AB_2758477), microtubule-associated proteins 1A/1B light chain 3B (LC3; rabbit, 1:1000, Abcam, Cat# ab48394, RRID: AB_881433), endoplasmic reticulum chaperone BiP (Grp78/BIP; mouse, 1:1000, Proteintech, Cat# 66574-1-Ig, RRID: AB_2881934), cyclic AMP-dependent transcription factor ATF-6 alpha (ATF6; rabbit, 1:1000, Proteintech, Cat# 24169-1-AP, RRID: AB_2876891), PRKR-like endoplasmic reticulum kinase (PERK; rabbit, 1:1000, Cell Signaling Technology, Cat# 3192S, RRID: AB_2095847), phospho-PERK (rabbit, 1:1000, Cell Signaling Technology, Cat# 3179S, RRID: AB_2095853), eukaryotic translation initiation factor 2 subunit alpha (eIF2α; rabbit, 1:1000, Cell Signaling Technology, Cat# 9722S, RRID: AB_2230924), phospho-eIF2α (rabbit, 1:1000, Cell Signaling Technology, 9721S, RRID: AB_330951), phosphatidylinositol 3-kinase regulatory subunit alpha (PI3 kinase p85; rabbit, 1:1000, Cell Signaling Technology, Cat# 4257S, RRID: AB_659889), RAC-alpha serine/threonine-protein kinase (AKT; rabbit, 1:1000, Cell Signaling Technology, Cat# 4691S, RRID: AB_915783), phospho-AKT (rabbit, 1:1000, Cell Signaling Technology, Cat# 4060S, RRID: AB_2315049), mammalian target of rapamycin (mTOR; rabbit, 1:1000, Cell Signaling Technology, Cat# 2983S, RRID: AB_2105622), phospho-mTOR (rabbit, 1:1000, Cell Signaling Technology, Cat# 2971S, RRID: AB_330970), and β-actin (mouse, 1:1000, Proteintech, Cat# 66009-1-Ig, RRID: AB_2687938).

    Techniques: In Vitro, Control, Immunofluorescence, Staining, Labeling, Recombinant

    The PI3K inhibitor LY294002 reverses the anti-ER stress effect of rtPA and the EGF domain of rtPA may mediate the PI3K/AKT pathway in the ICH in vitro cell model. (A–C) Analysis of ER stress–associated proteins ( n = 3 per group). (D) Confocal images and three-dimensional reconstruction of endoplasmic reticulum continuity of neurons by ER tracker after rtPA and PI3K inhibitor LY294002 treatment. Scale bars: 3 µm. (E) Immunofluorescence images of p-PERK (red, labeled by Cy3) in neurons after rtPA and PI3K inhibitor LY294002 treatment. Scale bars: 50 µm. (F–H) Analysis of PI3K p85 and p-AKT. Data are represented as mean ± SEM ( n = 3 per group). * P < 0.05, ** P < 0.01, *** P < 0.001, vs . hemin group; & P < 0.05, && P < 0.01, vs . hemin + rtPA group; # P < 0.05, vs . hemin + rtPA + DMSO group (one-way analysis of variance followed by Tukey’s post hoc test). (I) Transmission electron microscopy images of cells after rtPA and rtPA domain inhibitor treatment. Scale bar: 100 µm. 3D: Three-dimensional; AKT: RAC-alpha serine/threonine-protein kinase; ATF6: cyclic AMP-dependent transcription factor ATF-6 alpha; DAPI: 4′,6-diamidino-2-phenylindole; EGF: epidermal growth factor; eIF2α: eukaryotic translation initiation factor 2 subunit alpha; ER: endoplasmic reticulum; LY294002: PI3K inhibitor; mTOR: mammalian target of rapamycin; PERK: PRKR-like endoplasmic reticulum kinase; PI3K: phosphatidylinositol 3-kinase regulatory subunit alpha; rtPA: recombinant tissue plasminogen activator.

    Journal: Neural Regeneration Research

    Article Title: Recombinant tissue plasminogen activator protects neurons after intracerebral hemorrhage through activating the PI3K/AKT/mTOR pathway

    doi: 10.4103/NRR.NRR-D-23-01953

    Figure Lengend Snippet: The PI3K inhibitor LY294002 reverses the anti-ER stress effect of rtPA and the EGF domain of rtPA may mediate the PI3K/AKT pathway in the ICH in vitro cell model. (A–C) Analysis of ER stress–associated proteins ( n = 3 per group). (D) Confocal images and three-dimensional reconstruction of endoplasmic reticulum continuity of neurons by ER tracker after rtPA and PI3K inhibitor LY294002 treatment. Scale bars: 3 µm. (E) Immunofluorescence images of p-PERK (red, labeled by Cy3) in neurons after rtPA and PI3K inhibitor LY294002 treatment. Scale bars: 50 µm. (F–H) Analysis of PI3K p85 and p-AKT. Data are represented as mean ± SEM ( n = 3 per group). * P < 0.05, ** P < 0.01, *** P < 0.001, vs . hemin group; & P < 0.05, && P < 0.01, vs . hemin + rtPA group; # P < 0.05, vs . hemin + rtPA + DMSO group (one-way analysis of variance followed by Tukey’s post hoc test). (I) Transmission electron microscopy images of cells after rtPA and rtPA domain inhibitor treatment. Scale bar: 100 µm. 3D: Three-dimensional; AKT: RAC-alpha serine/threonine-protein kinase; ATF6: cyclic AMP-dependent transcription factor ATF-6 alpha; DAPI: 4′,6-diamidino-2-phenylindole; EGF: epidermal growth factor; eIF2α: eukaryotic translation initiation factor 2 subunit alpha; ER: endoplasmic reticulum; LY294002: PI3K inhibitor; mTOR: mammalian target of rapamycin; PERK: PRKR-like endoplasmic reticulum kinase; PI3K: phosphatidylinositol 3-kinase regulatory subunit alpha; rtPA: recombinant tissue plasminogen activator.

    Article Snippet: The following primary antibodies were used for analysis: bcl2 (rabbit, 1:1000, Proteintech, Cat# 12789-1-AP, RRID: AB_2227948), bax (rabbit, 1:1000, Proteintech, Cat# 50599-2-Ig, RRID: AB_2061561), coiled-coil myosin-like bcl2-interacting protein (beclin1; rabbit, 1:1000, Proteintech, Cat# 11306-1-AP, RRID: AB_2259061), sequestosome-1/ubiquitin-binding protein p62 (SQSTM1/p62; rabbit, 1:1000, Abclonal, Cat# A11250, RRID: AB_2758477), microtubule-associated proteins 1A/1B light chain 3B (LC3; rabbit, 1:1000, Abcam, Cat# ab48394, RRID: AB_881433), endoplasmic reticulum chaperone BiP (Grp78/BIP; mouse, 1:1000, Proteintech, Cat# 66574-1-Ig, RRID: AB_2881934), cyclic AMP-dependent transcription factor ATF-6 alpha (ATF6; rabbit, 1:1000, Proteintech, Cat# 24169-1-AP, RRID: AB_2876891), PRKR-like endoplasmic reticulum kinase (PERK; rabbit, 1:1000, Cell Signaling Technology, Cat# 3192S, RRID: AB_2095847), phospho-PERK (rabbit, 1:1000, Cell Signaling Technology, Cat# 3179S, RRID: AB_2095853), eukaryotic translation initiation factor 2 subunit alpha (eIF2α; rabbit, 1:1000, Cell Signaling Technology, Cat# 9722S, RRID: AB_2230924), phospho-eIF2α (rabbit, 1:1000, Cell Signaling Technology, 9721S, RRID: AB_330951), phosphatidylinositol 3-kinase regulatory subunit alpha (PI3 kinase p85; rabbit, 1:1000, Cell Signaling Technology, Cat# 4257S, RRID: AB_659889), RAC-alpha serine/threonine-protein kinase (AKT; rabbit, 1:1000, Cell Signaling Technology, Cat# 4691S, RRID: AB_915783), phospho-AKT (rabbit, 1:1000, Cell Signaling Technology, Cat# 4060S, RRID: AB_2315049), mammalian target of rapamycin (mTOR; rabbit, 1:1000, Cell Signaling Technology, Cat# 2983S, RRID: AB_2105622), phospho-mTOR (rabbit, 1:1000, Cell Signaling Technology, Cat# 2971S, RRID: AB_330970), and β-actin (mouse, 1:1000, Proteintech, Cat# 66009-1-Ig, RRID: AB_2687938).

    Techniques: In Vitro, Immunofluorescence, Labeling, Transmission Assay, Electron Microscopy, Recombinant