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Description
In unstimulated HeLa cells, GalNac-T1 (gray) and Helix Pomatia Lectin (HPL) (green) colocalize exclusively at the Giantin-stained Golgi apparatus (red). The Tn antigen refers to terminal α-linked N-acetyl galactosamine residues (GalNAc) linked to Ser or Thr residues. HPL binds various glycans but the Tn antigen in particular. HeLa cells were serum starved overnight in DME (noFBS). Cells were fixed for 10 min (4% paraformaldehyde) and permeabilized (0.2% Triton X-100). Primary antibody staining followed the manufacturer's instructions. Cells were subsequently stained for 15–30 min with secondary Alexa Fluor–conjugated antibodies (Alexa 488 for anti-Giantin, Alexa 594 for anti-GalNac-T1). Hoechst (blue) and Alexa 647-conjugated-HPL were added during secondary antibody incubations. Cells were mounted onto glass slides using FluorSave (Merck) and imaged at room temperature using an inverted FluoView confocal microscope (model IX81; Olympus) with fluorescence excitation at 488 nm, 561 nm, and 633 nm and either a 60x objective (U Plan Super Apochromatic [UPLSAPO]; NA 1.35) or 100x objective (UPLSAPO; NA 1.40) using Immersol oil. Microscope coupled with a CCD camera (model FVII). Images were acquired and processed using Olympus FV10-ASW software. The amount of GalNac-T1 (blue) staining or HPL staining (green) colocalizing with Golgi membranes (red) was compared between unstimulated and 4h EGF-treated HeLa cells using fixed laser power and detector voltages. The quantification is graphed in Fig2D. Image corresponds to Fig 2C, top panel, unstimulated, in J Cell Biol. 189: 843-858. 2010. Images in Fig 2 include CIL#s 13547, 13548, 13549, 13550. Research Data Curation Program, UC San Diego, La Jolla, 92093-0175 (https://lib.ucsd.edu/rdcp) Gill, David J.; Chia, Joanne; Senewiratne, Jamie; Bard, Frederic (2021). CIL:13549, Homo sapiens, epithelial cell, cervical carcinoma. In Cell Image Library. UC San Diego Library Digital Collections. Dataset. https://doi.org/10.6075/J0KW5DSV
Type
image
Identifier
ark:/20775/bb20544107
Language
No linguistic content
Subject
N-acetylgalactosamine binding HeLa Manganese ion binding Protein O-linked glycosylation via serine Protein O-linked glycosylation via threonine Golgi organization Polypeptide N-acetylgalactosaminyltransferase activity Integral to membrane Epithelial cell Golgi stack Cervical carcinoma Nucleus Golgi cisterna membrane Homo sapiens Cell Image Library Group ID: 6490
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