Helix Pomatia Lectin (HPL) (green) staining is redistributed out of the Golgi of HeLa cells after EGF treatment for 4 h, while the Golgi marker Giantin (red) is unaffected. 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. Image also includes anti-GalNacT1 staining (gray). 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 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. Image corresponds to Fig 1B in J Cell Biol. 189: 843-858. 2010. Images in Fig 1 include CIL#s 13543, 13544, 13545, 13546.