Journal: Journal of cellular physiology

Article Title: Identification of RUNX3 as a Component of the MST/Hpo Signaling Pathway

doi: 10.1002/jcp.22887

Figure Lengend Snippet: RUNX3 is a component of the MST pathway. A: A human fetal liver cDNA library was screened using a yeast two-hybrid system with full-length RUNX3 as bait. From 2 × 106 transformants, 32 positive clones encoding eight different genes were identified. Sequence analysis revealed that seven clones encoded CBFβ, a heterodimeric partner of RUNX3, and one of the positive clones encoded SAV1 (aa 51-260). Runt and WW indicate the Runt domain and the WW domain, respectively. B: HEK293 cells were transfected with a fixed amount of HA-tagged RUNX3 (0.5 μg) and increasing amounts of Flag-tagged SAV1 (0, 0.1, 0.2, and 0.4 μg), and were analyzed by immunoprecipitation (IP) with an anti-Flag antibody and by immunoblotting (IB) with anti-Flag or anti-HA antibodies. The expression levels of the transfected genes were detected by IB using the appropriate antibodies. Co-immunoprecipitated SAV1 and RUNX3 are indicated as RX3/SAV1. C: MST2 stimulated the interaction between RUNX3 and SAV1. HEK293 cells were transfected with an increasing amount of Myc-MST2 (0, 0.1, and 0.3 μg) and fixed amounts of HA-RUNX3 (0.5 μg)andFlag-SAV1(0.5 μg) as indicated. The association between SAV1 and RUNX3 was analyzed by IP with an anti-Flag antibody followed by IB with the appropriate antibodies. D: Endogenous RUNX3 and SAV1 formed a complex when MST2 was exogenously expressed. Lysates were obtained from HEK293 cells transfected with MST2 or vector, and the physical interaction between the endogenous RUNX3 and SAV1 was measured by IP with anti-SAV1 antibody followed by IB with anti-RUNX3 antibody (5G4). E: MST2 stimulated the interaction between SAV1 and endogenous RUNX3. Myc-SAV1 and HA-MST2 were expressed in HEK293 cells, and the interaction between exogenous SAV1 and endogenous RUNX3 was measured by IP (Myc) followed by IB with anti-RUNX3 antibody. Expression of Myc-SAV1, HA-MST2 and endogenous RUNX3 was detected by IB with the corresponding antibodies. F: MST2 interacted with RUNX3 through SAV1. HA-MST2, Myc-SAV1, and Flag-RUNX3 were expressedinHEK-293 cells as indicated, and the physical interaction between RUNX3 and MST2 was analyzed by IP followed by IB (top panel). The RUNX3–MST2 interaction was detected only when SAV1 was co-expressed.

Article Snippet: A human fetal liver cDNA library was screened as suggested in the manufacturer’s manual (OriGene).

Techniques: cDNA Library Assay, Clone Assay, Sequencing, Transfection, Immunoprecipitation, Western Blot, Expressing, Plasmid Preparation

Journal: iScience

Article Title: Akkermansia muciniphila induces mitochondrial calcium overload and α -synuclein aggregation in an enteroendocrine cell line

doi: 10.1016/j.isci.2022.103908

Figure Lengend Snippet:

Article Snippet: Human fetal brain cDNA library , Clontech , Cat# HL3003a.

Techniques: cDNA Library Assay, Recombinant, Proliferation Assay, Plasmid Preparation, Software