hes1  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Notch mediates non-regenerative alveolar repair after staphylococcal lung injury

doi: 10.64898/2026.02.04.703850

Figure Lengend Snippet: (A-D) Confocal images show immunofluorescence of GFP ( magenta ), DAPI ( cyan ), and E-cadherin ( yellow ) in cultured alveolar epithelial-like MLE-12 cells transfected with plasmid DNA encoding a Notch TMD-GFP fusion protein ( TMD-GFP ). Arrowheads in C-D point out locations where GFP and E-cadherin fluorescences merge, signaling TMD-GFP protein localizes to plasma membranes. Scale bars: 20 μm. (E) Immunoblot data show Hes1 and actin protein content in MLE-12 cells transfected with plasmid DNA encoding GFP or TMD-GFP, then either untreated or exposed to purified Hla ( Hla WT ) as indicated. Hla WT exposure was for 10 min, then the cells were incubated in culture media for 45 min prior to immunoblot processing. Immunoblots were each replicated 2x. (F-L) The cartoon in F shows the experimental design of the studies used to generate the data shown in G-L. As indicated in F, we carried out immunofluorescence determinations of E-cadherin expression ( yellow ) by confocal microscopy in MLE-12 cells transfected with plasmid DNA encoding GFP or TMD-GFP. Transfected cells were exposed to Hla WT as indicated. Note, while cells in both groups show a cell junctional pattern of E-cadherin under baseline conditions that is lost in response to Hla WT exposure, only TMD-GFP-transfected cells show E-cadherin recovery. Fluorescence of recovered E-cadherin in GFP-transfected cells appears disorganized and is not located at cell-cell junctions. Images each replicated 2x. Scale bars: 50 μm.

Article Snippet: Antibodies used for immunoblot studies included rabbit polyclonal antibody against NICD1 (MilliporeSigma; Anti-Notch1, NT; 1:500 dilution; cat. no. 07-1232, lot 3899517), rabbit mAb against Hes1 (Cell Signaling; clone D6P2U; 1:1000 dilution; cat. no. 11988, lot 4), and rabbit polyclonal antibody against actin (MilliporeSigma; 1:2000 dilution; cat. no. A2066, lot 202986).

Techniques: Immunofluorescence, Cell Culture, Transfection, Plasmid Preparation, Clinical Proteomics, Western Blot, Purification, Incubation, Expressing, Confocal Microscopy, Fluorescence