Journal: Biology Direct

Article Title: The MST4–14-3-3ζ complex promotes pancreatic cancer by activating YAP

doi: 10.1186/s13062-026-00784-6

Figure Lengend Snippet: MST4 Directly Binds to 14-3-3ζ via a Phosphorylation-dependent manner. ( A ). Left: Confocal microscope analysis of endogenous 14-3-3ζ and overexpressed Flag-MST4 in PANC-1 cells. DAPI represents the nuclear region. Scale bar: 10 μm. Right: Quantitative colocalization of MST4 and 14-3-3ζ in PANC-1 cells. Red pixel intensity presents Flag-MST4; Green pixel intensity presents 14-3-3ζ; Pearson r = 0.7441 ( n = 40). Colocalization quantification was performed using Pearson’s correlation coefficient analysis, based on methods reported in the literature . ( B ). Co-IP and immunoblot analysis of the interaction between exogenous Flag-tagged MST4 and 14-3-3ζ in HEK293FT cells. ( C ). MBP pulldown analysis between MST4/pMST4 and 14-3-3ζ. Purified protein of MST4 was first dephosphorylated by λPPase or further phosphorylated by ATP. The input and output samples were loaded on SDS-PAGE followed by CBB and immunoblot staining. ( D ). Microscale thermophoresis (MST) assay showing the binding affinity between 14-3-3ζ and the pMST4 (pT320) peptide. ( E ). Alignment of 14-3-3ζ binding motif sequence and the putative phosphorylation sites of MST4. The phosphorylation sites were labelled red. ( F ). Co-IP and immunoblot analysis of the interaction between exogenous Flag-tagged wildtype or T320A mutant of MST4 and endogenous 14-3-3ζ in HEK293FT cells

Article Snippet: HEK293FT cells were obtained from Shanghai Life Academy of Sciences cell library (Shanghai, China), and PANC-1 and KPC cells were purchased from Shanghai Model Organisms Center, Inc and Shanghai YuanChuang biotechnology Co.,Ltd (Shanghai, China), respectively.

Techniques: Phospho-proteomics, Microscopy, Co-Immunoprecipitation Assay, Western Blot, Purification, SDS Page, Staining, Microscale Thermophoresis, Binding Assay, Sequencing, Mutagenesis