Journal: Cell reports

Article Title: Hypoxic activation of PFKFB4 in breast tumor microenvironment shapes metabolic and cellular plasticity to accentuate metastatic competence

doi: 10.1016/j.celrep.2022.111756

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Sections were then processed with TrueVIEW Autofluorescence Quenching Kit (Vector Laboratories, Cat. No. SP-8400) to reduce the background following the manufacturer’s instruction and mounted in Anti-fade Mounting Medium with DAPI (Vector Laboratories, Cat. No. H-1800).

Techniques: Cell Culture, Produced, Recombinant, Infection, Western Blot, Lysis, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Plasmid Preparation, Blocking Assay, Stripping, Magnetic Beads, SYBR Green Assay, Luciferase, Amplification, Sequencing, shRNA, Software, Real-time Polymerase Chain Reaction

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Soluble polysialylated NCAM: a novel player of the innate immune system in the lung

doi: 10.1007/s00018-013-1342-0

Figure Lengend Snippet: The secretion of intracellularly localized polySia-NCAM of human lung epithelial cell line A549 is induced by IL-1β and LPS. a Western-blot analyses of cell lysates of human A549 cells were performed using a mAb against polySia and NCAM (123C3) before and after endoN treatment. b, c Cultured A549 cells were fixed with methanol and stained with fluorescently labeled inactive endoN and b anti-NCAM mAb 123C3 or c polyclonal antibodies to visualize the trans-Golgi apparatus (anti-TGN38). For nuclear staining DAPI was used. d In addition, polySia was visualized in cells after stimulation with IL-1β (200 ng/ml) or LPS (100 ng/ml). Scale bars for c and d = 25 μm. e, f Polysialylated proteins were purified from supernatants of differently treated A549 cells and visualized by Western blotting using an anti-polySia mAb and after depolysialylation with a mAb against NCAM (123C3). g In an analogous cell lysates of IL-1β-treated cells were analyzed. h Western blots against polySia and NCAM (after depolysialylation) were performed using supernatants of untreated A549 cells as well LPS-stimulated cells in the absence or presence of TAPI-2. Apparent molecular masses of standard proteins are indicated in kDa

Article Snippet: For immunostaining against polySia, NCAM, and TGN38, cells were fixed with methanol at −20 °C for 10 min, blocked with 1 % BSA in PBS for 15 min, and incubation with appropriately diluted primary antibodies or biotinylated inactive endoN was continued for 1 h. After three washing steps with PBS and exposure to streptavidin–FITC conjugate and an anti-mouse-Rhodamine secondary antibody (Dianova, Hamburg, Germany), the final mounting VECTASHIELD with DAPI (Linaris, Munich, Germany) was added to the fixed cells.

Techniques: Western Blot, Cell Culture, Staining, Labeling, Purification