yap1 ser109  (Cell Signaling Technology Inc)

Journal: International Journal of Molecular Sciences

Article Title: Calciprotein Particles Cause Endothelial Dysfunction under Flow

doi: 10.3390/ijms21228802

Figure Lengend Snippet: CPPs may impair endothelial mechanotransduction. ( A ) Western blotting measurements of mechanosensitive transcription factors KLF2, KLF4, NRF2, YAP1 and TAZ and phosphorylated forms of YAP1 and TAZ as compared to the expression of vimentin, CD31 and VE-cadherin in HCAEC and HITAEC co-incubated with PBS, MPP, CPP-P or CPP-S in a flow system for 4 h. Blot scans (left) and band densitometry analysis (right). The results of the latter are represented by a heat map. Green, gray and red colours mean fold change ≤ 0.75, 0.76–1.24, and ≥1.25, respectively, compared to PBS group; ( B ) gene expression analysis of KLF2 , KLF4 , NFE2L2 , YAP1 , and WWTR1 genes in HCAEC and HITAEC co-incubated with PBS, MPP, CPP-P or CPP-S in a flow system for 4 h. RT-qPCR measurements, the results are represented by a heat map. Gray and red colours mean fold change 0.51–1.99, and ≥2.00, respectively, compared to PBS group; ( C ) Western blotting measurements of mechanosensitive transcription factors Klf2, Klf4, Nrf2, Yap1, Taz and phosphorylated forms of Yap1 and Taz as compared to the expression of Cd31, Gapdh and histone H3 in the endothelial lysate collected from the descending aorta and aortic arch of Wistar rats which received consecutive tail vein injections of CPP-P, CPP-S or 0.9% NaCl (10 daily injections). Blot scans (left) and band densitometry analysis (right). The results of the latter are represented by a heat map. Green, gray and red colours mean fold change ≤ 0.75, 0.76–1.24, and ≥1.25, respectively, compared to NaCl group; ( D ) gene expression analysis of Klf2 , Klf4 , Nfe2l2 , Yap1 , and Wwtr1 genes in the endothelial lysate collected from the descending aorta and aortic arch of Wistar rats which received consecutive tail vein injections of CPP-P, CPP-S or 0.9% NaCl (10 daily injections). RT-qPCR measurements, the results are represented by a heat map. Green, gray and red colours mean fold change ≤ 0.50, 0.51–1.99, and ≥2.00, respectively, compared to the NaCl group. HCAEC—human coronary artery endothelial cells, HITAEC—human internal thoracic artery endothelial cells, PBS—phosphate-buffered saline, MPP—magnesiprotein particles, CPP-P—primary calciprotein particles, CPP-S—secondary calciprotein particles, KLF—Krüppel-like factor, NRF—nuclear factor erythroid 2–related factor, YAP—Yes-associated protein, pYAP—phosphorylated YAP, TAZ—transcriptional co-activator with PDZ binding motif, pTAZ—phosphorylated TAZ, CD31—cluster of differentiation 31, VE-cadherin—vascular endothelial cadherin, NFE2L2—nuclear factor erythroid 2 like 2, WWTR—WW domain containing transcription regulator, Gapdh—glyceraldehyde 3-phosphate dehydrogenase, RT-qPCR—reverse transcription quantitative polymerase chain reaction.

Article Snippet: Blots were probed with rabbit antibodies to VCAM1 (ab134047, 1:1000, Abcam, Cambridge, UK), ICAM1 (ab109361, 1:1000, Abcam, Cambridge, UK), Snail and Slug (ab180714, 1:500, Abcam, Cambridge, UK), KLF4 (ab215036, 1:200, Abcam, Cambridge, UK), NRF2 (ab62352, 1:200, Abcam, Cambridge, UK), YAP1 (14074, 1:500, Cell Signaling Technology, Danvers, MA, USA), phospho-YAP1-Ser109 (46931, 1:500, Cell Signaling Technology, Danvers, MA, USA), phospho-YAP1-Ser127 (13008, 1:500, Cell Signaling Technology, Danvers, MA, USA), phospho-YAP1-Ser397 (13619, 1:500, Cell Signaling Technology, Danvers, MA, USA), TAZ (70148, 1:500, Cell Signaling Technology, Danvers, MA, USA), phospho-TAZ-Ser89 (59971, 1:200, Cell Signaling Technology, Danvers, MA, USA), vimentin (loading control, ab16700, 1:500, Abcam, Cambridge, UK) and VE-cadherin (loading control, 361900, 1:100, Thermo Fisher Scientific, Waltham, MA, USA), or mouse antibodies to N-cadherin (MA515633, 1:500, Thermo Fisher Scientific, Waltham, MA, USA), TWIST1 (sc-81417, 1:100, Santa Cruz Biotechnology, Dallas, TX, USA), KLF2 (NBP2-61812, 1:200, Novus Biologicals, Littleton, CO, USA), CD31 (loading control, ab9498, 1:1000, Abcam, Cambridge, UK), and GAPDH/histone H3 (loading control, ab139416, 1:250, Abcam, Cambridge, UK).

Techniques: Western Blot, Expressing, Incubation, Gene Expression, Quantitative RT-PCR, Saline, Binding Assay, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Calciprotein Particles Cause Endothelial Dysfunction under Flow

doi: 10.3390/ijms21228802

Figure Lengend Snippet: Sequences of customised primers for RT-qPCR.

Article Snippet: Blots were probed with rabbit antibodies to VCAM1 (ab134047, 1:1000, Abcam, Cambridge, UK), ICAM1 (ab109361, 1:1000, Abcam, Cambridge, UK), Snail and Slug (ab180714, 1:500, Abcam, Cambridge, UK), KLF4 (ab215036, 1:200, Abcam, Cambridge, UK), NRF2 (ab62352, 1:200, Abcam, Cambridge, UK), YAP1 (14074, 1:500, Cell Signaling Technology, Danvers, MA, USA), phospho-YAP1-Ser109 (46931, 1:500, Cell Signaling Technology, Danvers, MA, USA), phospho-YAP1-Ser127 (13008, 1:500, Cell Signaling Technology, Danvers, MA, USA), phospho-YAP1-Ser397 (13619, 1:500, Cell Signaling Technology, Danvers, MA, USA), TAZ (70148, 1:500, Cell Signaling Technology, Danvers, MA, USA), phospho-TAZ-Ser89 (59971, 1:200, Cell Signaling Technology, Danvers, MA, USA), vimentin (loading control, ab16700, 1:500, Abcam, Cambridge, UK) and VE-cadherin (loading control, 361900, 1:100, Thermo Fisher Scientific, Waltham, MA, USA), or mouse antibodies to N-cadherin (MA515633, 1:500, Thermo Fisher Scientific, Waltham, MA, USA), TWIST1 (sc-81417, 1:100, Santa Cruz Biotechnology, Dallas, TX, USA), KLF2 (NBP2-61812, 1:200, Novus Biologicals, Littleton, CO, USA), CD31 (loading control, ab9498, 1:1000, Abcam, Cambridge, UK), and GAPDH/histone H3 (loading control, ab139416, 1:250, Abcam, Cambridge, UK).

Techniques: Sequencing