GnT1IP mislocalizes GlcNAcT-I-HA. (A) Myc-GnT1IP-L and -S were transiently coexpressed with GlcNAcT-I-HA in HeLa cells, fixed, immunolabeled for Myc (red) and for HA (green), and examined by confocal microscopy. Bars 10 µm. (B) Quantitation of effects of transiently expressed Myc-GnT1IP-L, Myc-GnT1Ip-S, or Myc-GnT1IP-S-CD1 on the localization of GlcNAcT-I-HA stably expressed in Lec1 cells. Localization in ∼300 cells per condition was classified into ER (E), more ER than Golgi (E/G), more Golgi than ER (G/E), or Golgi (G), and compared with Lec1 cells stably expressing GlcNAcT-I-HA alone. Error bars = SD; n = 3. (C) Deletion mutants of GnT1IP were coexpressed with GlcNAcT-I-HA in HeLa cells, fixed, immunolabeled for Myc (red) and for HA (green), and examined by immunofluoresence (top) or confocal microscopy (bottom). (D) Myc-GnT1IP-L was transiently expressed in HeLa cells, fixed, immunolabeled for Myc-GnT1IP-L (red) and endogenous ManII or β4GalT-I (green), and observed by fluorescence or confocal microscopy. (E) Myc-GnT1IP-L was either transiently expressed alone or coexpressed with GlcNAcT-I-HA in HeLa cells overnight, CHX was added for 1 h, followed by CHX and BFA for 30 min, cells were fixed, immunolabeled for Myc-GnT1IP-L (red) and GlcNAcT-I-HA or endogenous ManII (green), and observed by fluorescence microscopy. White boxes in the middle panel are enlarged below. Bars, 10 µm.