An ER-trapped GalNac-T activity reporter (Muc-PTS; fused to GFP) (green) is targeted to the ER and not the Golgi, marked by Giantin (red) in HeLa cells cotransfected with inactive Src (SrcK-mCherry, containing K295M mutation) (gray). 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 647 for anti-Giantin and Hoechst (blue). 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 5C in J Cell Biol. 189: 843-858. 2010. Images in Fig 5 include CIL#s 13559, 13560, 13561, 13562.
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