rabbit anti p yap1  (Cell Signaling Technology Inc)

Journal: Oncology Reports

Article Title: Epigenetic silencing of JAM3 promotes laryngeal squamous cell carcinoma development by inhibiting the Hippo pathway

doi: 10.3892/or.2024.8861

Figure Lengend Snippet: JAM3 mediates laryngeal squamous cell carcinoma tumorigenesis through the Hippo pathway. (A) Protein levels of Flag, JAM3, p-LATS1 (Thr1079), LATS1, p-YAP1 (Ser127), YAP1 and β-actin in AMC-HN-8 and FD-LSC-1 cells with overexpression or knockdown of JAM3 were detected by western blotting. (B) Expression of YAP1 in AMC-HN-8 and FD-LSC-1 cells after transfection with the JAM3 overexpression plasmid or si-JAM3 were detected by confocal microscopy. Scale bar, 50 µm. Fluorescence ratio of YAP1 in the cytoplasm and nucleus of (C) AMC-HN-8 and (D) FD-LSC-1 cells was calculated by ImageJ and analyzed by two-tailed Student's t-test or one-way ANOVA. Data are presented as the mean ± SD of three independent experiments. *P<0.05 vs. Vec; # P<0.05, ## P<0.01, ### P<0.001, #### P<0.0001 vs. si-NC group. JAM3, junctional adhesion molecule 3; LATS1, large tumor suppressor kinase 1; NC, negative control; p-, phosphorylated; si, small interfering; Vec, p3×Flag-CMV-10 empty vector; YAP1, yes-associated protein 1.

Article Snippet: The membranes were then blocked in 10% non-fat milk (BD Biosciences) for 1.5 h at room temperature to prevent non-specific binding, and were incubated with primary antibodies targeting Flag (cat. no. F1804; 1:1,000; mouse; MilliporeSigma), JAM3 (cat. no. bs-11086R; 1:1,000; rabbit; BIOSS), large tumor suppressor kinase 1 (LATS1; cat. no. 17049-1-AP; 1:1,000; rabbit; Proteintech Group, Inc.), phosphorylated (p)-LATS1 (Thr1079) (cat. no. 28998-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), yes-associated protein 1 (YAP1; cat. no. 13584-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), p-YAP1 (Ser127) (cat. no. 13008S; 1:1,000; rabbit; Cell Signaling Technology, Inc.) and β-actin (cat. no. HC201-02; 1:2,000; mouse; TransGen Biotech Co., Ltd.) overnight at 4°C.

Techniques: Over Expression, Knockdown, Western Blot, Expressing, Transfection, Plasmid Preparation, Confocal Microscopy, Fluorescence, Two Tailed Test, Negative Control

Journal: Oncology Reports

Article Title: Epigenetic silencing of JAM3 promotes laryngeal squamous cell carcinoma development by inhibiting the Hippo pathway

doi: 10.3892/or.2024.8861

Figure Lengend Snippet: Knockdown of JAM3 promotes tumorigenicity of laryngeal squamous cell carcinoma cells in vivo . (A) Representative images of tumors in nude mice after subcutaneous injection of AMC-HN-8 cells transfected with si-NC and si-JAM3. (B) Tumor growth curve was plotted using xenograft tumor volume data. ***P<0.001 vs. si-NC. (C) Tumor weight was measured after tumor excision. *P<0.05 vs. si-NC group. (D) Relative expression levels of JAM3 in xenograft tumors as determined by reverse transcription-quantitative PCR analysis. Data are presented as the mean ± SD of three independent experiments. ****P<0.0001 vs. si-NC. (E) Hematoxylin and eosin staining of xenograft tumors. Immunohistochemistry staining of (F) JAM3, Ki-67 and YAP1, and (G) E-cadherin, N-cadherin and Vimentin in xenograft tumors. Scale bar, 20 µm. JAM3, junctional adhesion molecule 3; NC, negative control; si, small interfering; YAP1, yes-associated protein 1.

Article Snippet: The membranes were then blocked in 10% non-fat milk (BD Biosciences) for 1.5 h at room temperature to prevent non-specific binding, and were incubated with primary antibodies targeting Flag (cat. no. F1804; 1:1,000; mouse; MilliporeSigma), JAM3 (cat. no. bs-11086R; 1:1,000; rabbit; BIOSS), large tumor suppressor kinase 1 (LATS1; cat. no. 17049-1-AP; 1:1,000; rabbit; Proteintech Group, Inc.), phosphorylated (p)-LATS1 (Thr1079) (cat. no. 28998-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), yes-associated protein 1 (YAP1; cat. no. 13584-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), p-YAP1 (Ser127) (cat. no. 13008S; 1:1,000; rabbit; Cell Signaling Technology, Inc.) and β-actin (cat. no. HC201-02; 1:2,000; mouse; TransGen Biotech Co., Ltd.) overnight at 4°C.

Techniques: Knockdown, In Vivo, Injection, Transfection, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Staining, Immunohistochemistry, Negative Control

Journal: Oncology Reports

Article Title: Epigenetic silencing of JAM3 promotes laryngeal squamous cell carcinoma development by inhibiting the Hippo pathway

doi: 10.3892/or.2024.8861

Figure Lengend Snippet: Mechanism by which epigenetic silencing of JAM3 promotes LSCC development by inhibiting the Hippo pathway. JAM3, junctional adhesion molecule 3; LATS1, large tumor suppressor kinase 1; YAP1, yes-associated protein 1; p, phosphorylated; EMT, epithelial-mesenchymal transition.

Article Snippet: The membranes were then blocked in 10% non-fat milk (BD Biosciences) for 1.5 h at room temperature to prevent non-specific binding, and were incubated with primary antibodies targeting Flag (cat. no. F1804; 1:1,000; mouse; MilliporeSigma), JAM3 (cat. no. bs-11086R; 1:1,000; rabbit; BIOSS), large tumor suppressor kinase 1 (LATS1; cat. no. 17049-1-AP; 1:1,000; rabbit; Proteintech Group, Inc.), phosphorylated (p)-LATS1 (Thr1079) (cat. no. 28998-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), yes-associated protein 1 (YAP1; cat. no. 13584-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), p-YAP1 (Ser127) (cat. no. 13008S; 1:1,000; rabbit; Cell Signaling Technology, Inc.) and β-actin (cat. no. HC201-02; 1:2,000; mouse; TransGen Biotech Co., Ltd.) overnight at 4°C.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Urolithin A attenuates hexavalent chromium-induced small intestinal injury by modulating PP2A/Hippo/YAP1 pathway

doi: 10.1016/j.jbc.2024.107669

Figure Lengend Snippet: The inv olvement of Hippo/YAP1 signaling pathway in the intestinal injury induced by Cr(VI). A , representative panoramic images of immunofluorescence staining with an antibody against YAP1 ( green ) in small intestinal sections from WT and HE mice treated with 20 mg/l Cr(VI) (40× and 200× magnification). The nuclei ( blue ) were stained with 4,6-diamino-2-phenyl indole (DAPI). B , YAP1 expression was quantified and expressed as relative fluorescence intensity normalized by to DAPI density. C , protein levels of indicated protein in small intestinal tissues were determined by immunoblotting analysis. D , representative images shown for small intestinal sections stained with p-YAP1(Ser127) antibody (200× magnification). E , quantification was shown for the fold change of indicated protein level relative to WT control mice. F , quantification of p-YAP1(Ser127)-stained area was expressed as percentage of small intestinal fields occupied (n = 3). ∗ p < 0.05, compared with WT control mice. # p < 0.05, compared with HE control mice. Cr(VI), hexavalent chromium; HE, heterozygous; PP2A, protein phosphatase 2A.

Article Snippet: The following antibodies were used: rabbit anti-PP2A Aα, anti-LATS1, anti-p-YAP1 (Ser109), p-YAP1 (Ser127) (Cell Signaling Technology), mouse anti-TAZ, rabbit anti-YAP1 (Proteintech group), and rabbit anti-PP2A Aβ (Bioworld).

Techniques: Immunofluorescence, Staining, Expressing, Fluorescence, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Urolithin A attenuates hexavalent chromium-induced small intestinal injury by modulating PP2A/Hippo/YAP1 pathway

doi: 10.1016/j.jbc.2024.107669

Figure Lengend Snippet: Hippo/YAP1 signaling pathway mediated Cr(VI)-induced intestinal injury by regulating cell proliferation and apoptosis. Representative images of staining with ( A ) Ki-67 and ( C ) TUNEL in small intestinal sections WT and HE mice upon 20 mg/l Cr(VI) treatment (200× magnification). The nuclei ( blue ) were stained with 4,6-diamino-2-phenyl indole. The numbers of ( B ) Ki-67–positive cells and ( D ) apoptotic cells were quantified and expressed as the average number of positive cells every five visual filed per mouse. Spearman correlation analysis was performed to analyze the association between YAP1 expression and the number of ( E ) Ki-67–positive cells or ( F ) apoptotic cells, respectively. ∗ p < 0.05, compared with WT control mice. # p < 0.05, compared with HE control mice. Cr(VI), hexavalent chromium; HE, heterozygous; TUNEL, terminal-deoxynucleotidyl transferase-mediated nick end labeling.

Article Snippet: The following antibodies were used: rabbit anti-PP2A Aα, anti-LATS1, anti-p-YAP1 (Ser109), p-YAP1 (Ser127) (Cell Signaling Technology), mouse anti-TAZ, rabbit anti-YAP1 (Proteintech group), and rabbit anti-PP2A Aβ (Bioworld).

Techniques: Staining, TUNEL Assay, Expressing, Control, End Labeling

Journal: The Journal of Biological Chemistry

Article Title: Urolithin A attenuates hexavalent chromium-induced small intestinal injury by modulating PP2A/Hippo/YAP1 pathway

doi: 10.1016/j.jbc.2024.107669

Figure Lengend Snippet: Urolithin A attenuated Cr(VI)-induced small intestinal injury by regulating Hippo/YAP1 signaling pathway. Mice were treated with 20 mg/l Cr(VI) in the presence or absence of urolithin A (20 mg/kg body weight) for 28 days (n = 10). A , representative panoramic images were shown for small intestinal sections stained with H&E (10× or 100× magnification). B , the length of intestinal villi (n = 3) and ( C ) depth of intestinal crypt (n = 3) were quantified by ImageJ. D , representative images were shown in small intestinal tissues stained with AB-PAS (200× magnification). E , AB-PAS staining was performed to count the number of goblet cells (n = 3). F , representative images of immunofluorescence staining with an antibody against occludin ( green ) in small intestinal tissues. The nuclei ( blue ) were stained with 4,6-diamino-2-phenyl indole. G , occludin expression was quantified and expressed as fluorescence intensity relative to control group (n = 3). H , the intestinal permeability was determined by the concentration of FITC-dextran in serum. I , the alkaline phosphatase (ALP) level was normalized to intestinal protein and expressed as unit/g protein. ∗ p < 0.05, compared with control mice. # p < 0.05, compared with urolithin A mice. AB-PAS, alcian blue/periodic acid-Schiff; Cr(VI), hexavalent chromium.

Article Snippet: The following antibodies were used: rabbit anti-PP2A Aα, anti-LATS1, anti-p-YAP1 (Ser109), p-YAP1 (Ser127) (Cell Signaling Technology), mouse anti-TAZ, rabbit anti-YAP1 (Proteintech group), and rabbit anti-PP2A Aβ (Bioworld).

Techniques: Staining, Immunofluorescence, Expressing, Fluorescence, Control, Permeability, Concentration Assay