After EGF stimulation for 4 h, Helix Pomatia Lectin (HPL) (green) in HeLa cells expressing wild-type Arf1-GFP (Arf1(WT)-GFP) (gray) is redistributed from Golgi (Giantin) (red) to ER. HPL binds various glycans but the Tn antigen in particular. The Tn antigen refers to terminal α-linked N-acetyl galactosamine residues (GalNAc) linked to Ser or Thr residues. HPL indicates distribution of GalNac-Ts. HeLa cells were serum starved overnight in DME (noFBS) and then treated with human recombinant EGF (100 ng/ml; Sigma-Aldrich) for 4h. 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 594 for anti-Giantin. 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. Image corresponds to Fig 7B, left 2 panels in J Cell Biol. 189: 843-858. 2010. Images in Fig 7 include CIL#s 13571, 13572, 13573, 13574, 13575, 13576, 13577.
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