HeLa cells expressing Myc-GnT1IP-L (Myc-tagged GlcNAcT-1 inhibitory protein, long transcript, membrane-anchored) (red) and GlcNAcT-1-HA (green) were treated with cycloheximide and brefeldin A to cause retrograde transport of Golgi enzymes into the ER. Both proteins had punctate costaining around the nucleus, typical of ERGIC localization, in addition to reticular costaining of the ER after BFA treatment. Transiently expressed Myc-GnT1IP-L is normally primarily localized in the Golgi and ERGIC and partially in the ER (shown in Fig 4; J Cell Biol. 190: 893-910, 2010.). GlcNAcT-1 is normally a resident of the medial Golgi. Transfected HeLa cells were fixed in 3% paraformaldehyde, blocked with 0.2% Triton X-100, 1% FBS, 0.5% BSA in PBS with Ca[2+] and Mg[2+], followed by incubation in primary antibodies (anti-Myc mouse mAb, 9E10 (Covance) and anti–HA mouse mAb, HA.11 (Covance)) and secondary antibodies (Alexa 568 and Alexa 488) and DAPI (blue) to stain nuclei. Cells were mounted using Fluoromount (SouthernBiotech). Immunofluorescent images were digitally acquired on an inverted microscope (Axiovert 200M; Carl Zeiss, Inc.) coupled to a 12-bit cooled charge-coupled device camera (AxioCam MRm Rev. 3; Carl Zeiss, Inc.) controlled by Axiovision software (AxioVs40, version 22.214.171.124; Carl Zeiss, Inc.), using a 100x/1.3 NA oil immersion objective (EC Plan-NeoFluar; Carl Zeiss, Inc.), and saved as tiff files (1388 × 1040, 8 bit). All pictures were treated identically using Adobe Photoshop to remove background and adjust contrast. This image corresponds to Figure 5E, middle panel (merge) in J Cell Biol. 190: 893-910, 2010. Images in Fig 5 include CIL#s 13658, 24919, 24920.
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