p merlin s518 rabbit cell signaling technology 9163 ip  (Cell Signaling Technology Inc)

Journal: Environmental health and preventive medicine

Article Title: Lead exposure promotes NF2-wildtype meningioma cell proliferation through the Merlin-Hippo signaling pathway.

doi: 10.1265/ehpm.24-00216

Figure Lengend Snippet: Fig. 4 Merlin expression in meningioma cells after lead exposure. (A). Detection of Merlin protein in IOMM-Lee and CH157-MN cells. (B) Detection of Merlin and phosphorylated Merlin (Ser518) after lead treatment in IOMM-Lee cells. (C) Quantification of relative expression levels of Merlin normalized to GAPDH. (D) Quantification of relative expression levels of p-Merlin (Ser518) normalized to total Merlin. Data represents the mean + SEM. Statistical difference was determined using one-way ANOVA test with n = 3–8 samples per group (* p < 0.05; ** p < 0.01).

Article Snippet: Nuclear protein and cytoplasmic protein extraction kit (P0028) was from China Beyotime Inc. Merlin antibody (#9168), p-Merlin (Ser518) antibody (#13281), MST1 antibody (#14946), p-MST1(Thr183)/MST 2(Thr180) antibody (#49332), LATS antibody (#3477), YAP/TAZ antibody (#8418), YAP antibody (#14074), p-YAP(Ser127) antibody (#13008), and Lamin B1 antibody (#13435) purchased from CST, USA.

Techniques: Expressing

Journal: iScience

Article Title: JAM-A signals through the Hippo pathway to regulate intestinal epithelial proliferation

doi: 10.1016/j.isci.2022.104316

Figure Lengend Snippet: JAM-A regulates proliferation and associates with Hippo regulatory molecules LATS1 and NF2 (A) JAM-A-deficient mice exhibit increased colonic IEC proliferation. Representative confocal images of colon tissue from JAM-A ERΔIEC and control ( JAM-A fl/fl ) mice stained for JAM-A (green), Ki67 (red), and DAPI/nuclei (blue). Increased levels of proliferating cells in the JAM-A ERΔIEC mice compared with controls is shown in the graph plotting percentage of Ki67 positive cells/crypt with dots denoting individual crypts. Data are means ± SEM of crypts from JAM-A fl/fl and JAM-A ERΔIEC mice, respectively. ∗∗∗p ≤ 0.001; two-tailed Student’s t test. Scale bar, 50 μm. (B) EdU incorporation in 3D enteroids derived from JAM-A −/− and WT ( JAM-A +/+ ) mice indicate that loss of JAM-A results in increased proliferation. Data are means ± SEM where buds counted from WT and JAM-A −/− enteroids and are denoted as dots on the graph. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001: two-tailed Student’s t test. Scale bar, 50 μm. (C) Co-immunoprecipitation of ZO-2 and JAM-A with NF2. Whole-cell lysate of SKCO-15 IEC stably expressing a full-length HA-tagged JAM-A (HA-JAM-A) was immunoprecipitated with a ZO-2 (ZO-2 IP) or HA (HA IP) antibody followed by immunoblotting for NF2, ZO-2 and NF2, p-NF2 S518, and HA-JAM-A. p-NF2 S518 did not co-immunoprecipitate with HA-JAM-A. (D) Representative immunostaining of NF2 (green) and JAM-A (red) detected by confocal microscopy in WT murine colon tissue demonstrate co-localization at the TJ. Scale bar, 50 μm. (E) JAM-A co-immunoprecipitates with multiple Hippo pathway molecules. Immunoprecipitation of HA-tagged JAM-A from IEC lysates with an HA-specific antibody (HA IP) followed by western blot LATS1, YAP, p-YAP S127, 14-3-3, and JAM-A. Co-immunoprecipitation of these molecules with JAM-A is demonstrated. (F) Endogenous JAM-A co-immunoprecipitation with LATS1, NF2, and YAP. Immunoprecipitation of LATS1 with LATS1 antibody (LATS1 IP) from IEC cell lysates followed by western blot for NF2, YAP, JAM-A, and LATS1. Co-immunoprecipitation is observed between these molecules. (G) Representative immunofluorescence confocal images of LATS1 (green), JAM-A (red), and DAPI/nuclei (blue) in WT murine colon tissue. Scale bar, 50 μm. Lysate blots serve as input loading controls in (C), (E), and (F). Immunoprecipitation with a nonspecific IgG antibody served as a negative control (IgG IP) for (C), (E), and (F). All data are representative of three independent experiments. See also Figure S1 .

Article Snippet: Rabbit Anti-p-NF2/Merlin S518 , Cell Signaling Technology , 9163; RRID: AB_2149793.

Techniques: Control, Staining, Two Tailed Test, Derivative Assay, Immunoprecipitation, Stable Transfection, Expressing, Western Blot, Immunostaining, Confocal Microscopy, Immunofluorescence, Negative Control

Journal: iScience

Article Title: JAM-A signals through the Hippo pathway to regulate intestinal epithelial proliferation

doi: 10.1016/j.isci.2022.104316

Figure Lengend Snippet: JAM-A is a regulator of Hippo signaling in primary cultured IEC (A) Representative confocal immunostaining of NF2 (green) and JAM-A (red) in WT murine 2D colonoids shows co-localization at the TJ. Scale bar, 20 μm. (B) Representative confocal immunostaining of LATS1 (green) and JAM-A (red) associating at the TJ in WT 2D murine colonoids. Scale bar, 20 μm. (C) JAM-A ERΔIEC primary IEC exhibit prolonged nuclear YAP localization. Representative immunofluorescent images of YAP (green) and ZO-1 (red) in 2D colonoids cultured at low and high cell confluency derived from and JAM-A ERΔIEC and JAM-A fl/fl mice. At low cell confluency YAP staining is visible in the nuclei of JAM-A ERΔIEC and JAM-A fl/fl IEC. At high cell confluency, YAP localizes to the cytoplasm in control IEC but remains in the nuclei of JAM-A ERΔIEC IEC, revealing a failure of Hippo signaling initiation. Graph depicts scoring of YAP localization (nuclear, cytoplasm, and nuclear + cytoplasm). Five fields of view for each condition were scored to generate an average percentage, and three independent experiments were performed (denoted by the dots). Scale bar, 20 μm. (D) JAM-A-deficient primary IEC exhibit decreased Hippo signaling activity. Representative western blots for p-LATS1 S909, LATS1, p-NF2 S518, NF2, p-YAP S127, YAP, and JAM-A in subconfluent 2D colonoids derived from JAM-A ERΔIEC and JAM-A fl/f l mice. Graphs represent digital densitometry for relative density of p-NF2 S518, p-LATS1 S909, and p-YAP S127. JAM-A ERΔIEC colonoids exhibit decreased NF2 and LATS1 activity, resulting in increased YAP activity relative to JAM-A fl/f l controls. Calnexin serves as a loading control. Data are means ± SEM of three independent experiments. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001: two-tailed Student’s t test.

Article Snippet: Rabbit Anti-p-NF2/Merlin S518 , Cell Signaling Technology , 9163; RRID: AB_2149793.

Techniques: Cell Culture, Immunostaining, Derivative Assay, Staining, Control, Activity Assay, Western Blot, Two Tailed Test

Journal: iScience

Article Title: JAM-A signals through the Hippo pathway to regulate intestinal epithelial proliferation

doi: 10.1016/j.isci.2022.104316

Figure Lengend Snippet:

Article Snippet: Rabbit Anti-p-NF2/Merlin S518 , Cell Signaling Technology , 9163; RRID: AB_2149793.

Techniques: Recombinant, Plasmid Preparation, SYBR Green Assay, DC Protein Assay, Imaging, Derivative Assay, Sequencing, Negative Control, Software, Microscopy, Spectrophotometry

Journal: Aging (Albany NY)

Article Title: NF2 deficiency accelerates neointima hyperplasia following vascular injury via promoting YAP-TEAD1 interaction in vascular smooth muscle cells

doi: 10.18632/aging.103240

Figure Lengend Snippet: NF2 knockdown enhances neointima hyperplasia after vascular injury. WT or Nf2 -/- mice received sham operation or wire injury in common carotid artery. ( A ) The relative protein levels of p-NF2 Ser518 and NF2 in common carotid arteries from C57BL/6J mice at day 7, 14 and 28 after injury were analyzed by immunoblotting (n=5). ( B ) The relative mRNA (n=5) level of Nf2 in common carotid arteries from WT or Nf2 -/- mice. ( C ) The relative protein (n=3) levels of p-NF2 Ser518 and NF2 in common carotid arteries from WT or Nf2 -/- mice. ( D ) Representative H&E staining of carotid arteries from WT or Nf2 -/- mice at day 28 after sham operation or wire injury (left) and corresponding quantification for ratio of intima/media (right) were shown (n=6). Magnification 200×. ( E ) Immunohistochemistry staining of Ki-67 (brown) in sections of carotid arteries from WT or Nf2 -/- mice at day 28 after sham operation or wire injury (left) and corresponding quantification for Ki-67 positive cells within neointima (right) were shown (n=6). Magnification 200×. Data are shown as mean ± S.D. * P <0.05, ** P <0.01 and *** P <0.001 denote statistical comparison between the two marked groups, respectively.

Article Snippet: For Ki-67, p-NF2 Ser518 and NF2 staining, the artery sections were firstly incubated with primary antibody against Ki-67, p-NF2 Ser518 and NF2 (Cell Signaling Technology, Danvers, MA) at 4°C overnight after being blocked, followed by incubation with secondary antibody and finally counterstained with mayer hematoxylin.

Techniques: Knockdown, Western Blot, Staining, Immunohistochemistry, Comparison

Journal: Aging (Albany NY)

Article Title: NF2 deficiency accelerates neointima hyperplasia following vascular injury via promoting YAP-TEAD1 interaction in vascular smooth muscle cells

doi: 10.18632/aging.103240

Figure Lengend Snippet: VSMC proliferation and migration in vitro is elevated after NF2 knockdown. ( A ) The relative protein expression levels of p-NF2 Ser518 and NF2 were determined by immunoblotting in VSMC after 0, 24 and 48 h of physiological saline or PDGF-BB (30 ng/mL) treatment (n=5). ( B ) VSMC isolated from WT or Nf2 -/- mice was stained with SM α-actin (red), Ki-67 (green) and DAPI (blue) after 48 h of physiological saline or PDGF-BB (30 ng/mL) treatment. Representative images (left) and corresponding quantification of Ki-67 positive VSMC (right) were shown (n=5). Magnification 400×. ( C ) Migration of VSMC isolated from WT and Nf2 -/- mice after 24 h of physiological saline or PDGF-BB (30 ng/mL) treatment was measured via wound healing assay. Representative images (upper panel) and corresponding quantification of healing rates (lower panel) were shown (n=5). Magnification 100×. ( D ) VSMC isolated from WT or Nf2 -/- mice after 8 h of physiological saline or PDGF-BB (30 ng/mL) treatment was assessed by transwell assay. Representative images (upper panel) and corresponding quantification of migration cells (lower panel) were shown (n=5). Magnification 100×. Data are shown as mean ± S.D. ** P <0.01 and *** P <0.001 denote statistical comparison between the two marked groups, respectively.

Article Snippet: For Ki-67, p-NF2 Ser518 and NF2 staining, the artery sections were firstly incubated with primary antibody against Ki-67, p-NF2 Ser518 and NF2 (Cell Signaling Technology, Danvers, MA) at 4°C overnight after being blocked, followed by incubation with secondary antibody and finally counterstained with mayer hematoxylin.

Techniques: Migration, In Vitro, Knockdown, Expressing, Western Blot, Saline, Isolation, Staining, Wound Healing Assay, Transwell Assay, Comparison

Journal: Aging (Albany NY)

Article Title: NF2 deficiency accelerates neointima hyperplasia following vascular injury via promoting YAP-TEAD1 interaction in vascular smooth muscle cells

doi: 10.18632/aging.103240

Figure Lengend Snippet: Enhanced NF2 phosphorylation and subsequent nuclear translocation of YAP following PDGF-BB treatment or injury. ( A ) Nuclear and cytosolic-enriched fractions were prepared from VSMC, which was treated by PDGF-BB (30 ng/mL) for 48 h. The relative protein expression levels of p-NF2 Ser518 , NF2 and YAP were determined by immunoblotting (n=5). ( B ) Nuclear and cytosolic-enriched fractions were prepared from arteries of C57BL/6J mice at day 28 after vascular injury. The relative protein expression levels of p-NF2 Ser518 , NF2 and YAP were determined by immunoblotting (n=5). Data are shown as mean ± S.D. ** P <0.01 and *** P <0.001 denote statistical comparison between the two marked groups, respectively.

Article Snippet: For Ki-67, p-NF2 Ser518 and NF2 staining, the artery sections were firstly incubated with primary antibody against Ki-67, p-NF2 Ser518 and NF2 (Cell Signaling Technology, Danvers, MA) at 4°C overnight after being blocked, followed by incubation with secondary antibody and finally counterstained with mayer hematoxylin.

Techniques: Phospho-proteomics, Translocation Assay, Expressing, Western Blot, Comparison

Journal: Aging (Albany NY)

Article Title: NF2 deficiency accelerates neointima hyperplasia following vascular injury via promoting YAP-TEAD1 interaction in vascular smooth muscle cells

doi: 10.18632/aging.103240

Figure Lengend Snippet: NF2 knockdown accelerates PDGF-BB-induced VSMC proliferation and migration in a YAP-dependent manner. VSMC isolated from WT or Nf2 -/- mice was transfected by shCon, shYap, Ad-Con and Ad-Yap. Then the VSMC received 48 h of physiological saline or PDGF-BB (30 ng/mL) treatment. ( A ) The relative protein expression levels of p-YAP Ser127 and YAP were determined by immunoblotting in VSMC treated as above mentioned (n=5). ( B ) VSMC treated as above mentioned was stained with SM α-actin (red), Ki-67 (green) and DAPI (blue). Representative images (left) and corresponding quantification of Ki-67-positive VSMC (right) were shown (n=5). Magnification 400×. ( C ) VSMC treated as above mentioned was assessed by transwell assay. Representative images (left) and corresponding quantification of migration cells (right) were shown (n=5). Magnification 100×. Data are shown as mean ± S.D. * P <0.05, ** P <0.01 and *** P <0.001 denote statistical comparison between the two marked groups, respectively.

Article Snippet: For Ki-67, p-NF2 Ser518 and NF2 staining, the artery sections were firstly incubated with primary antibody against Ki-67, p-NF2 Ser518 and NF2 (Cell Signaling Technology, Danvers, MA) at 4°C overnight after being blocked, followed by incubation with secondary antibody and finally counterstained with mayer hematoxylin.

Techniques: Knockdown, Migration, Isolation, Transfection, Saline, Expressing, Western Blot, Staining, Transwell Assay, Comparison

Journal: Aging (Albany NY)

Article Title: NF2 deficiency accelerates neointima hyperplasia following vascular injury via promoting YAP-TEAD1 interaction in vascular smooth muscle cells

doi: 10.18632/aging.103240

Figure Lengend Snippet: YAP is required for NF2 knockdown-mediated neointima hyperplasia after vascular injury. WT and Nf2 -/- mice received injection of shCon, shYap, Ad-Con and Ad-Yap into the injured left common carotid artery via the external carotid artery immediately after injury and then incubated for 30 min. The mice subsequently received intravenous injection of these adenovirus via tail vein at 7, 14, 21 days after injury. ( A ) The relative protein expression levels of p-YAP Ser127 and YAP were determined by immunoblotting in arteries of WT and Nf2 -/- mice at day 28 after vascular injury (n=5). ( B ) Representative H&E staining of carotid arteries from WT or Nf2 -/- mice treated as above mentioned (left) and corresponding quantification for ratio of intima/media (right) were shown (n=5). Magnification 200×. ( C ) Immunohistochemistry staining of Ki-67 (brown) in sections of carotid arteries from WT or Nf2 -/- mice treated as above mentioned (left) and corresponding quantification for Ki-67-positive cells within neointima (right) were shown (n=5). Magnification 200×. Data are shown as mean ± S.D. * P <0.05, ** P <0.01 and *** P <0.001 denote statistical comparison between the two marked groups, respectively.

Article Snippet: For Ki-67, p-NF2 Ser518 and NF2 staining, the artery sections were firstly incubated with primary antibody against Ki-67, p-NF2 Ser518 and NF2 (Cell Signaling Technology, Danvers, MA) at 4°C overnight after being blocked, followed by incubation with secondary antibody and finally counterstained with mayer hematoxylin.

Techniques: Knockdown, Injection, Incubation, Expressing, Western Blot, Staining, Immunohistochemistry, Comparison

Journal: Aging (Albany NY)

Article Title: NF2 deficiency accelerates neointima hyperplasia following vascular injury via promoting YAP-TEAD1 interaction in vascular smooth muscle cells

doi: 10.18632/aging.103240

Figure Lengend Snippet: NF2 knockdown causes increased VSMC proliferation and migration induced by PDGF-BB via inducing YAP-TEAD1 interaction. ( A ) VSMC isolated from WT or Nf2 -/- mice after 48 h of physiological saline or PDGF-BB (30 ng/mL) treatment was subjected to immunoprecipitation using anti-YAP antibody or control IgG. Inputs and immunocomplexes were analyzed by immunoblotting. VSMC isolated from WT and Nf2 -/- mice was transfected with control siRNA, Teadi or Ad-Con, Ad-Yap and then treated by physiological saline or PDGF-BB (30 ng/mL) for 48 h. ( B ) VSMC treated as above mentioned was stained with SM α-actin (red), Ki-67 (green) and DAPI (blue). Representative images (left) and corresponding quantification of Ki-67-positive VSMC (right) were shown (n=5). Magnification 400×. ( C ) VSMC treated as above mentioned was assessed by transwell assay. Representative images (left) and corresponding quantification of migration cells (right) were shown (n=5). Magnification 100×. Data are shown as mean ± S.D. * P <0.05, ** P <0.01 and *** P <0.001 denote statistical comparison between the two marked groups, respectively.

Article Snippet: For Ki-67, p-NF2 Ser518 and NF2 staining, the artery sections were firstly incubated with primary antibody against Ki-67, p-NF2 Ser518 and NF2 (Cell Signaling Technology, Danvers, MA) at 4°C overnight after being blocked, followed by incubation with secondary antibody and finally counterstained with mayer hematoxylin.

Techniques: Knockdown, Migration, Isolation, Saline, Immunoprecipitation, Control, Western Blot, Transfection, Staining, Transwell Assay, Comparison

Journal: Aging (Albany NY)

Article Title: NF2 deficiency accelerates neointima hyperplasia following vascular injury via promoting YAP-TEAD1 interaction in vascular smooth muscle cells

doi: 10.18632/aging.103240

Figure Lengend Snippet: The hypothesis of NF2/YAP/TEAD1 signaling axis in VSMC after PDGF-BB stimuli. PDGF-BB excretion after vascular injury induces nuclear NF2 phosphorylation, thereby causing inactivation of NF2. Decreased active NF2 led to dephosphorylation of YAP and subsequent nuclear translocation of YAP. Increased nuclear YAP binds to TEAD1 and positively regulates relative target gene expression.

Article Snippet: For Ki-67, p-NF2 Ser518 and NF2 staining, the artery sections were firstly incubated with primary antibody against Ki-67, p-NF2 Ser518 and NF2 (Cell Signaling Technology, Danvers, MA) at 4°C overnight after being blocked, followed by incubation with secondary antibody and finally counterstained with mayer hematoxylin.

Techniques: Phospho-proteomics, De-Phosphorylation Assay, Translocation Assay, Targeted Gene Expression

Journal: Aging (Albany NY)

Article Title: NF2 deficiency accelerates neointima hyperplasia following vascular injury via promoting YAP-TEAD1 interaction in vascular smooth muscle cells

doi: 10.18632/aging.103240

Figure Lengend Snippet: NF2 knockdown enhances neointima hyperplasia after vascular injury. WT or Nf2 -/- mice received sham operation or wire injury in common carotid artery. ( A ) The relative protein levels of p-NF2 Ser518 and NF2 in common carotid arteries from C57BL/6J mice at day 7, 14 and 28 after injury were analyzed by immunoblotting (n=5). ( B ) The relative mRNA (n=5) level of Nf2 in common carotid arteries from WT or Nf2 -/- mice. ( C ) The relative protein (n=3) levels of p-NF2 Ser518 and NF2 in common carotid arteries from WT or Nf2 -/- mice. ( D ) Representative H&E staining of carotid arteries from WT or Nf2 -/- mice at day 28 after sham operation or wire injury (left) and corresponding quantification for ratio of intima/media (right) were shown (n=6). Magnification 200×. ( E ) Immunohistochemistry staining of Ki-67 (brown) in sections of carotid arteries from WT or Nf2 -/- mice at day 28 after sham operation or wire injury (left) and corresponding quantification for Ki-67 positive cells within neointima (right) were shown (n=6). Magnification 200×. Data are shown as mean ± S.D. * P <0.05, ** P <0.01 and *** P <0.001 denote statistical comparison between the two marked groups, respectively.

Article Snippet: Antibodies against phospho (p)-NF2 Ser518 , NF2, p-YAP Ser127 , YAP, TEAD1, GAPDH, H3, β-actin were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Knockdown, Western Blot, Staining, Immunohistochemistry, Comparison

Journal: Aging (Albany NY)

Article Title: NF2 deficiency accelerates neointima hyperplasia following vascular injury via promoting YAP-TEAD1 interaction in vascular smooth muscle cells

doi: 10.18632/aging.103240

Figure Lengend Snippet: VSMC proliferation and migration in vitro is elevated after NF2 knockdown. ( A ) The relative protein expression levels of p-NF2 Ser518 and NF2 were determined by immunoblotting in VSMC after 0, 24 and 48 h of physiological saline or PDGF-BB (30 ng/mL) treatment (n=5). ( B ) VSMC isolated from WT or Nf2 -/- mice was stained with SM α-actin (red), Ki-67 (green) and DAPI (blue) after 48 h of physiological saline or PDGF-BB (30 ng/mL) treatment. Representative images (left) and corresponding quantification of Ki-67 positive VSMC (right) were shown (n=5). Magnification 400×. ( C ) Migration of VSMC isolated from WT and Nf2 -/- mice after 24 h of physiological saline or PDGF-BB (30 ng/mL) treatment was measured via wound healing assay. Representative images (upper panel) and corresponding quantification of healing rates (lower panel) were shown (n=5). Magnification 100×. ( D ) VSMC isolated from WT or Nf2 -/- mice after 8 h of physiological saline or PDGF-BB (30 ng/mL) treatment was assessed by transwell assay. Representative images (upper panel) and corresponding quantification of migration cells (lower panel) were shown (n=5). Magnification 100×. Data are shown as mean ± S.D. ** P <0.01 and *** P <0.001 denote statistical comparison between the two marked groups, respectively.

Article Snippet: Antibodies against phospho (p)-NF2 Ser518 , NF2, p-YAP Ser127 , YAP, TEAD1, GAPDH, H3, β-actin were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Migration, In Vitro, Knockdown, Expressing, Western Blot, Saline, Isolation, Staining, Wound Healing Assay, Transwell Assay, Comparison

Journal: Aging (Albany NY)

Article Title: NF2 deficiency accelerates neointima hyperplasia following vascular injury via promoting YAP-TEAD1 interaction in vascular smooth muscle cells

doi: 10.18632/aging.103240

Figure Lengend Snippet: Enhanced NF2 phosphorylation and subsequent nuclear translocation of YAP following PDGF-BB treatment or injury. ( A ) Nuclear and cytosolic-enriched fractions were prepared from VSMC, which was treated by PDGF-BB (30 ng/mL) for 48 h. The relative protein expression levels of p-NF2 Ser518 , NF2 and YAP were determined by immunoblotting (n=5). ( B ) Nuclear and cytosolic-enriched fractions were prepared from arteries of C57BL/6J mice at day 28 after vascular injury. The relative protein expression levels of p-NF2 Ser518 , NF2 and YAP were determined by immunoblotting (n=5). Data are shown as mean ± S.D. ** P <0.01 and *** P <0.001 denote statistical comparison between the two marked groups, respectively.

Article Snippet: Antibodies against phospho (p)-NF2 Ser518 , NF2, p-YAP Ser127 , YAP, TEAD1, GAPDH, H3, β-actin were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Phospho-proteomics, Translocation Assay, Expressing, Western Blot, Comparison

Journal: Aging (Albany NY)

Article Title: NF2 deficiency accelerates neointima hyperplasia following vascular injury via promoting YAP-TEAD1 interaction in vascular smooth muscle cells

doi: 10.18632/aging.103240

Figure Lengend Snippet: NF2 knockdown accelerates PDGF-BB-induced VSMC proliferation and migration in a YAP-dependent manner. VSMC isolated from WT or Nf2 -/- mice was transfected by shCon, shYap, Ad-Con and Ad-Yap. Then the VSMC received 48 h of physiological saline or PDGF-BB (30 ng/mL) treatment. ( A ) The relative protein expression levels of p-YAP Ser127 and YAP were determined by immunoblotting in VSMC treated as above mentioned (n=5). ( B ) VSMC treated as above mentioned was stained with SM α-actin (red), Ki-67 (green) and DAPI (blue). Representative images (left) and corresponding quantification of Ki-67-positive VSMC (right) were shown (n=5). Magnification 400×. ( C ) VSMC treated as above mentioned was assessed by transwell assay. Representative images (left) and corresponding quantification of migration cells (right) were shown (n=5). Magnification 100×. Data are shown as mean ± S.D. * P <0.05, ** P <0.01 and *** P <0.001 denote statistical comparison between the two marked groups, respectively.

Article Snippet: Antibodies against phospho (p)-NF2 Ser518 , NF2, p-YAP Ser127 , YAP, TEAD1, GAPDH, H3, β-actin were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Knockdown, Migration, Isolation, Transfection, Saline, Expressing, Western Blot, Staining, Transwell Assay, Comparison

Journal: Aging (Albany NY)

Article Title: NF2 deficiency accelerates neointima hyperplasia following vascular injury via promoting YAP-TEAD1 interaction in vascular smooth muscle cells

doi: 10.18632/aging.103240

Figure Lengend Snippet: YAP is required for NF2 knockdown-mediated neointima hyperplasia after vascular injury. WT and Nf2 -/- mice received injection of shCon, shYap, Ad-Con and Ad-Yap into the injured left common carotid artery via the external carotid artery immediately after injury and then incubated for 30 min. The mice subsequently received intravenous injection of these adenovirus via tail vein at 7, 14, 21 days after injury. ( A ) The relative protein expression levels of p-YAP Ser127 and YAP were determined by immunoblotting in arteries of WT and Nf2 -/- mice at day 28 after vascular injury (n=5). ( B ) Representative H&E staining of carotid arteries from WT or Nf2 -/- mice treated as above mentioned (left) and corresponding quantification for ratio of intima/media (right) were shown (n=5). Magnification 200×. ( C ) Immunohistochemistry staining of Ki-67 (brown) in sections of carotid arteries from WT or Nf2 -/- mice treated as above mentioned (left) and corresponding quantification for Ki-67-positive cells within neointima (right) were shown (n=5). Magnification 200×. Data are shown as mean ± S.D. * P <0.05, ** P <0.01 and *** P <0.001 denote statistical comparison between the two marked groups, respectively.

Article Snippet: Antibodies against phospho (p)-NF2 Ser518 , NF2, p-YAP Ser127 , YAP, TEAD1, GAPDH, H3, β-actin were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Knockdown, Injection, Incubation, Expressing, Western Blot, Staining, Immunohistochemistry, Comparison

Journal: Aging (Albany NY)

Article Title: NF2 deficiency accelerates neointima hyperplasia following vascular injury via promoting YAP-TEAD1 interaction in vascular smooth muscle cells

doi: 10.18632/aging.103240

Figure Lengend Snippet: NF2 knockdown causes increased VSMC proliferation and migration induced by PDGF-BB via inducing YAP-TEAD1 interaction. ( A ) VSMC isolated from WT or Nf2 -/- mice after 48 h of physiological saline or PDGF-BB (30 ng/mL) treatment was subjected to immunoprecipitation using anti-YAP antibody or control IgG. Inputs and immunocomplexes were analyzed by immunoblotting. VSMC isolated from WT and Nf2 -/- mice was transfected with control siRNA, Teadi or Ad-Con, Ad-Yap and then treated by physiological saline or PDGF-BB (30 ng/mL) for 48 h. ( B ) VSMC treated as above mentioned was stained with SM α-actin (red), Ki-67 (green) and DAPI (blue). Representative images (left) and corresponding quantification of Ki-67-positive VSMC (right) were shown (n=5). Magnification 400×. ( C ) VSMC treated as above mentioned was assessed by transwell assay. Representative images (left) and corresponding quantification of migration cells (right) were shown (n=5). Magnification 100×. Data are shown as mean ± S.D. * P <0.05, ** P <0.01 and *** P <0.001 denote statistical comparison between the two marked groups, respectively.

Article Snippet: Antibodies against phospho (p)-NF2 Ser518 , NF2, p-YAP Ser127 , YAP, TEAD1, GAPDH, H3, β-actin were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Knockdown, Migration, Isolation, Saline, Immunoprecipitation, Control, Western Blot, Transfection, Staining, Transwell Assay, Comparison

Journal: Aging (Albany NY)

Article Title: NF2 deficiency accelerates neointima hyperplasia following vascular injury via promoting YAP-TEAD1 interaction in vascular smooth muscle cells

doi: 10.18632/aging.103240

Figure Lengend Snippet: The hypothesis of NF2/YAP/TEAD1 signaling axis in VSMC after PDGF-BB stimuli. PDGF-BB excretion after vascular injury induces nuclear NF2 phosphorylation, thereby causing inactivation of NF2. Decreased active NF2 led to dephosphorylation of YAP and subsequent nuclear translocation of YAP. Increased nuclear YAP binds to TEAD1 and positively regulates relative target gene expression.

Article Snippet: Antibodies against phospho (p)-NF2 Ser518 , NF2, p-YAP Ser127 , YAP, TEAD1, GAPDH, H3, β-actin were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Phospho-proteomics, De-Phosphorylation Assay, Translocation Assay, Targeted Gene Expression