After EGF treatment of HeLa cells for 4h, a significant amount of GalNac-T1 staining (gray) is apparent in punctate and diffuse cytoplasmic cellular structures that stain positively for Helix Pomatia Lectin (HPL) (green). Anti-Giantin marks the Golgi (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) 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. 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, bottom panel, 4h EGF-treated, in J Cell Biol. 189: 843-858. 2010. Images in Fig 2 include CIL#s 13547, 13548, 13549, 13550.