Journal: CNS Neuroscience & Therapeutics

Article Title: Asparagine Endopeptidase Inhibition Attenuates Tissue Plasminogen Activator‐Induced Brain Hemorrhagic Transformation After Ischemic Stroke

doi: 10.1111/cns.70345

Figure Lengend Snippet: AEP KO suppresses LRP‐1, MMP2, and MMP9 induced by delayed tPA treatment. (A, B) Immunofluorescence staining to assess the expression and location of AEP and CD31 (Scar bar = 100 μm). Data are presented as mean ± SEM, and statistical analysis is performed using a one‐way ANOVA test followed by Tukey's multiple comparisons test. (C, D) Western blotting to evaluate the expression of AEP, LRP‐1, MMP2, MMP3, and MMP9. Data are presented as mean ± SEM, and statistical analysis is performed using the Welch test followed by the Dunnett T3 multiple comparisons test were applied since the P value of the Levene test < 0.05. (A) n = 3 (3 sections and 9 areas), (C) n = 3 per group. Normality and variance are assessed via Shapiro‐Wilk test and Levene's test, respectively. * P < 0.05, ** P < 0.01, *** P < 0.001, ns: Not significant.

Article Snippet: The following antibodies and reagents were used: Legumain (AEP) (#93627, Cell Signaling Technology, 1:1000 for western blotting and 1:500 for immunofluorescence), ZO‐1 (YN1410, Immunoway, 1:1000 for western blotting), claudin‐5 (YT0953, Immunoway, 1:1000 for western blotting), occludin (YN2865, Immunoway, 1:1000 for western blotting), JAM‐1 (YT5479, Immunoway, 1:1000 for western blotting), Collagen IV (YM3756, Immunoway, 1:500 for immunofluorescence), CD31 (YM6115, Immunoway, 1:500 for immunofluorescence), LRP‐1 (#64099, Cell Signaling Technology, 1:500 for western blotting), MMP2 (YT2798, Immunoway, 1:1000 for western blotting), MMP3 (YT4465, Immunoway, 1:1000 for western blotting), MMP9 (YT1892, Immunoway, 1:1000 for western blotting), phospho‐TrkB (#4621, Cell Signaling Technology, 1:500 for western blotting), TrkB (#4603, Cell Signaling Technology, 1:500 for western blotting), β‐Actin (#4970, Cell Signaling Technology, 1:2000 for western blotting), anti‐rabbit IgG‐HRP (#70745, Cell Signaling Technology, 1:3000 for western blotting), Alexa Fluor 594‐conjugated goat anti‐mouse IgG (Invitrogen, A‐11005, 1:1000 for immunofluorescence), Alexa Fluor 488‐conjugated goat anti‐rabbit IgG (Invitrogen, A‐32731, 1:1000 for immunofluorescence), DAPI (C1002, Beyotime), TUNEL cell death detection kit (C1090, Beyotime), 2,3,5‐Triphenyl‐2H‐Tetrazolium Chloride (TTC) (G3005, Solarbio), Evans Blue Solution (E8010, Solarbio), Hematoxylin Staining Solution (C0170, Beyotime), Eosin Staining Solution (C0190, Beyotime), Cell Counting Kit‐8 (C0037, Beyotime).

Techniques: Immunofluorescence, Staining, Expressing, Western Blot

Journal: CNS Neuroscience & Therapeutics

Article Title: Asparagine Endopeptidase Inhibition Attenuates Tissue Plasminogen Activator‐Induced Brain Hemorrhagic Transformation After Ischemic Stroke

doi: 10.1111/cns.70345

Figure Lengend Snippet: AEP KO suppresses LRP‐1, MMP2, and MMP9 induced by delayed tPA treatment. (A, B) Immunofluorescence staining to assess the expression and location of AEP and CD31 (Scar bar = 100 μm). Data are presented as mean ± SEM, and statistical analysis is performed using a one‐way ANOVA test followed by Tukey's multiple comparisons test. (C, D) Western blotting to evaluate the expression of AEP, LRP‐1, MMP2, MMP3, and MMP9. Data are presented as mean ± SEM, and statistical analysis is performed using the Welch test followed by the Dunnett T3 multiple comparisons test were applied since the P value of the Levene test < 0.05. (A) n = 3 (3 sections and 9 areas), (C) n = 3 per group. Normality and variance are assessed via Shapiro‐Wilk test and Levene's test, respectively. * P < 0.05, ** P < 0.01, *** P < 0.001, ns: Not significant.

Article Snippet: The following antibodies and reagents were used: Legumain (AEP) (#93627, Cell Signaling Technology, 1:1000 for western blotting and 1:500 for immunofluorescence), ZO‐1 (YN1410, Immunoway, 1:1000 for western blotting), claudin‐5 (YT0953, Immunoway, 1:1000 for western blotting), occludin (YN2865, Immunoway, 1:1000 for western blotting), JAM‐1 (YT5479, Immunoway, 1:1000 for western blotting), Collagen IV (YM3756, Immunoway, 1:500 for immunofluorescence), CD31 (YM6115, Immunoway, 1:500 for immunofluorescence), LRP‐1 (#64099, Cell Signaling Technology, 1:500 for western blotting), MMP2 (YT2798, Immunoway, 1:1000 for western blotting), MMP3 (YT4465, Immunoway, 1:1000 for western blotting), MMP9 (YT1892, Immunoway, 1:1000 for western blotting), phospho‐TrkB (#4621, Cell Signaling Technology, 1:500 for western blotting), TrkB (#4603, Cell Signaling Technology, 1:500 for western blotting), β‐Actin (#4970, Cell Signaling Technology, 1:2000 for western blotting), anti‐rabbit IgG‐HRP (#70745, Cell Signaling Technology, 1:3000 for western blotting), Alexa Fluor 594‐conjugated goat anti‐mouse IgG (Invitrogen, A‐11005, 1:1000 for immunofluorescence), Alexa Fluor 488‐conjugated goat anti‐rabbit IgG (Invitrogen, A‐32731, 1:1000 for immunofluorescence), DAPI (C1002, Beyotime), TUNEL cell death detection kit (C1090, Beyotime), 2,3,5‐Triphenyl‐2H‐Tetrazolium Chloride (TTC) (G3005, Solarbio), Evans Blue Solution (E8010, Solarbio), Hematoxylin Staining Solution (C0170, Beyotime), Eosin Staining Solution (C0190, Beyotime), Cell Counting Kit‐8 (C0037, Beyotime).

Techniques: Immunofluorescence, Staining, Expressing, Western Blot

Journal: CNS Neuroscience & Therapeutics

Article Title: Asparagine Endopeptidase Inhibition Attenuates Tissue Plasminogen Activator‐Induced Brain Hemorrhagic Transformation After Ischemic Stroke

doi: 10.1111/cns.70345

Figure Lengend Snippet: AEP inhibitor 7,8‐DHF protect HUVECs against oxygen–glucose deprivation by inhibiting tPA‐induced elevated LRP‐1, MMP2, and MMP9. HUVECs treated with 0.5 μM 7,8‐DHF for 24 h followed by OGD 4 h and reoxygen‐glucose plus tPA (500 ng/mL) for another 24 h as the methods part described. (A, B) Western blotting to evaluate the expression of ZO‐1, claudin5, occluding, and JAM‐1. (C) The viability of HUVECs at different time points. (D) AEP enzymatic analysis of HUVECs subjected to 24 h tPA treatment following 7,8‐DHF and OGD. (E, F) Western blotting to evaluate the expression of AEP, p‐TrkB, TrkB, LRP‐1, MMP2, and MMP9. (A) n = 4, (C) n = 6, (D) n = 4, (E) n = 4 per group. Data are presented as mean ± SEM, and statistical analysis is performed using one‐way ANOVA test followed by Tukey's multiple comparisons test when P value of Levene test > 0.05 or Welch test followed by Dunnett T3 multiple comparisons test when the P value of Levene test < 0.05. Normality and variance are assessed via Shapiro‐Wilk test and Levene's test, respectively. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The following antibodies and reagents were used: Legumain (AEP) (#93627, Cell Signaling Technology, 1:1000 for western blotting and 1:500 for immunofluorescence), ZO‐1 (YN1410, Immunoway, 1:1000 for western blotting), claudin‐5 (YT0953, Immunoway, 1:1000 for western blotting), occludin (YN2865, Immunoway, 1:1000 for western blotting), JAM‐1 (YT5479, Immunoway, 1:1000 for western blotting), Collagen IV (YM3756, Immunoway, 1:500 for immunofluorescence), CD31 (YM6115, Immunoway, 1:500 for immunofluorescence), LRP‐1 (#64099, Cell Signaling Technology, 1:500 for western blotting), MMP2 (YT2798, Immunoway, 1:1000 for western blotting), MMP3 (YT4465, Immunoway, 1:1000 for western blotting), MMP9 (YT1892, Immunoway, 1:1000 for western blotting), phospho‐TrkB (#4621, Cell Signaling Technology, 1:500 for western blotting), TrkB (#4603, Cell Signaling Technology, 1:500 for western blotting), β‐Actin (#4970, Cell Signaling Technology, 1:2000 for western blotting), anti‐rabbit IgG‐HRP (#70745, Cell Signaling Technology, 1:3000 for western blotting), Alexa Fluor 594‐conjugated goat anti‐mouse IgG (Invitrogen, A‐11005, 1:1000 for immunofluorescence), Alexa Fluor 488‐conjugated goat anti‐rabbit IgG (Invitrogen, A‐32731, 1:1000 for immunofluorescence), DAPI (C1002, Beyotime), TUNEL cell death detection kit (C1090, Beyotime), 2,3,5‐Triphenyl‐2H‐Tetrazolium Chloride (TTC) (G3005, Solarbio), Evans Blue Solution (E8010, Solarbio), Hematoxylin Staining Solution (C0170, Beyotime), Eosin Staining Solution (C0190, Beyotime), Cell Counting Kit‐8 (C0037, Beyotime).

Techniques: Western Blot, Expressing

Journal: CNS Neuroscience & Therapeutics

Article Title: Asparagine Endopeptidase Inhibition Attenuates Tissue Plasminogen Activator‐Induced Brain Hemorrhagic Transformation After Ischemic Stroke

doi: 10.1111/cns.70345

Figure Lengend Snippet: R13 inhibits the activation of LRP‐1, MMP2, and MMP9 induced by delayed tPA treatment to ameliorate the destruction of tight junction proteins destruction. (A, B) Western blotting to evaluate the expression of ZO‐1, claudin5, occluding, JAM‐1. (C, D) Western blotting to evaluate the expression of AEP, p‐TrkB, TrkB, LRP‐1, MMP2, MMP9. (A), (C) n = 4 per group. Data are presented as mean ± SEM and statistical analyses are performed using one‐way ANOVA test followed by Tukey's multiple comparisons test when P value of Levene test > 0.05 or Welch test followed by Dunnett T3 multiple comparisons test when the P value of Levene test < 0.05. Normality and variance are assessed via Shapiro‐Wilk test and Levene's test, respectively. * P < 0.05, ** P < 0.05, *** P < 0.001.

Article Snippet: The following antibodies and reagents were used: Legumain (AEP) (#93627, Cell Signaling Technology, 1:1000 for western blotting and 1:500 for immunofluorescence), ZO‐1 (YN1410, Immunoway, 1:1000 for western blotting), claudin‐5 (YT0953, Immunoway, 1:1000 for western blotting), occludin (YN2865, Immunoway, 1:1000 for western blotting), JAM‐1 (YT5479, Immunoway, 1:1000 for western blotting), Collagen IV (YM3756, Immunoway, 1:500 for immunofluorescence), CD31 (YM6115, Immunoway, 1:500 for immunofluorescence), LRP‐1 (#64099, Cell Signaling Technology, 1:500 for western blotting), MMP2 (YT2798, Immunoway, 1:1000 for western blotting), MMP3 (YT4465, Immunoway, 1:1000 for western blotting), MMP9 (YT1892, Immunoway, 1:1000 for western blotting), phospho‐TrkB (#4621, Cell Signaling Technology, 1:500 for western blotting), TrkB (#4603, Cell Signaling Technology, 1:500 for western blotting), β‐Actin (#4970, Cell Signaling Technology, 1:2000 for western blotting), anti‐rabbit IgG‐HRP (#70745, Cell Signaling Technology, 1:3000 for western blotting), Alexa Fluor 594‐conjugated goat anti‐mouse IgG (Invitrogen, A‐11005, 1:1000 for immunofluorescence), Alexa Fluor 488‐conjugated goat anti‐rabbit IgG (Invitrogen, A‐32731, 1:1000 for immunofluorescence), DAPI (C1002, Beyotime), TUNEL cell death detection kit (C1090, Beyotime), 2,3,5‐Triphenyl‐2H‐Tetrazolium Chloride (TTC) (G3005, Solarbio), Evans Blue Solution (E8010, Solarbio), Hematoxylin Staining Solution (C0170, Beyotime), Eosin Staining Solution (C0190, Beyotime), Cell Counting Kit‐8 (C0037, Beyotime).

Techniques: Activation Assay, Western Blot, Expressing