pcdna flag yap1  (Addgene inc)

Journal: bioRxiv

Article Title: FZD2 inhibits YAP and prevents cell cycle reentry in adult murine cardiomyocytes

doi: 10.1101/2024.07.26.605158

Figure Lengend Snippet: A. Relative activity of TEAD-Gal4-UAS-Luc, a YAP reporter consisting of TEAD1 fused to the GAL4 DNA-binding domain and a UAS-driven firefly luciferase, in NVCMs transfected with empty vector or YAP1 expression plasmid and either control or FZD2 siRNA. B. Relative activity of the 8x-TEAD-Luc reporter, which uses 8 copies of the TEAD consensus binding site to express firefly luciferase, in NVCMs transfected with empty vector or YAP1-expression plasmid and control or FZD2 siRNA. C. TEAD-Gal4-UAS-Luc activity in NVCMs transfected with empty vector or YAP expression plasmid with or without FZD2 expression vector. D. TEAD-Gal4-UAS-Luc activity in NVCMs transfected with empty vector, YAP-expression vector, or expression plasmid for a YAP mutant with serine 127 mutated to alanine (YAP S127A) either alone or in combination with FZD2 expression vector. E. TEAD-Gal4-UAS-Luc activity in NVCMs transfected with empty vector, YAP expression plasmid, or YAP S127A expression vector and either control or FZD2 siRNA. F. TEAD-Gal4-UAS-Luc activity in NVCMs transfected with empty vector or YAP1 expression plasmid and FZD2 or β-catenin siRNA. G. TEAD-Gal4-UAS-Luc activity in NVCMs transfected with empty vector, YAP1 expression vector, FZD2 expression vector, or both FZD2 and YAP1 vectors as well as control or β-catenin siRNA. Graphs represent the mean ± S.D, asterisks indicate p ≤ 0.05 by two-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: NVCMs were also transfected with pRK5- mFzd2 and pcDNA-Flag- Yap1 (AddGene, 42254 and 18881, respectively) and pCMV-Tag2 (Agilent Technologies, 211172) for empty vector controls.

Techniques: Activity Assay, Binding Assay, Luciferase, Transfection, Plasmid Preparation, Expressing, Control, Mutagenesis

Journal: bioRxiv

Article Title: Crk mediates Csk-Hippo signaling independently of Yap tyrosine phosphorylation to induce cell extrusion

doi: 10.1101/2024.06.27.601065

Figure Lengend Snippet: (A) Cre-mediated depletion of Csk in MEF cells led to the loss of pSrc Y527 inhibitory phosphorylation and a concomitant increase in pSrc Y416 activating phosphorylation without perturbing ERK phosphorylation. (B) CRISPR-Cas9 genome editing was used to mutate two tyrosine phosphorylation sites ( Yap Y341F/Y357F or Yap Y2F ). (C) Luminal dilation and Mist1 expression defect were not rescued in Csk CKO ; Yap Y2F mutants. (D) Yap tyrosine phosphorylation was reduced but not lost in Yap Y2F homozygous MEF cells. (E) Mutating all three tyrosine residues in a Flag-tagged Yap (Yap Y3F ) led to a complete loss of tyrosine phosphorylation as revealed by Flag or phospho-tyrosine (pY) immunoprecipitation. (F) Schematic diagram of CRISPR-Cas9 genome editing to generate Yap Y341F/Y357F/Y394F ( Yap Y3F ) and Taz Y321F ( Taz YF ) mutant mice. (G) Yap protein remained nuclear in Csk CKO ; Yap Y3F ; Taz YF mutants, resulting in luminal dilation and loss of Mist1 expression. (H) The lumen size was quantified as the ratio of the lumen area versus the overall epithelial area. One-way ANOVA, n=5, *P<0.01. (I) Quantification of Mist1+ cells. One-way ANOVA, n=5, *P<0.0001. Scale bars: 100 µm.

Article Snippet: Plasmids encoding Yap 2YF and Yap 3YF were constructed from pcDNA Flag-Yap1 Y357F (Addgene #18882) through site-directed mutagenesis using Neb Q5 high-fidelity polymerase (New England Biolab #M0491).

Techniques: Phospho-proteomics, CRISPR, Expressing, Immunoprecipitation, Mutagenesis