Description

Helix Pomatia Lectin (HPL) (green) redistribution out of the Golgi (Giantin) (red) in HeLa cells upon EGF stimulation for 4 h is inhibited by co-treatement of Src Kinase Inhibitor, SKI. 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 treated with human recombinant EGF (100 ng/ml; Sigma-Aldrich) and 10 µM Src Kinase Inhibitor I; Merck. 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). 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 405 nm, 488 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 3A, bottom left, Src Inhibitor treated/4 hours EGF, in J Cell Biol. 189: 843-858. 2010. Images in Fig 3 include CIL#s 13551, 13552, 13553, 13554.