Published customer image:
Sheep anti Human TGN46 antibody used for the identification of the trans golgi network in MD-MB-231 cells by immunofluorescence.
Image caption:
APP redistributes from endosomes to the TGN upon overexpression of µ4-F255A-HA. MD-MB-231 cells were cotransfected with a plasmid encoding either of the indicated HA-epitope-tagged variants of μ4, and with a plasmid encoding APP-GFP carrying the double mutation F615P/D664A. After 24-h cells were fixed, permeabilized, stained for EEA1 and TGN46, and examined by fluorescence microscopy. Merging green, red, and blue channels generated the fourth image on each row; yellow indicates overlapping localization of the green and red channels, cyan indicates overlapping localization of the green and blue channels, magenta indicates overlapping localization of the red and blue channels, and white indicates overlapping localization of the red, green, and blue channels. Insets show 2× magnifications. Bar, 10 μm.

From: Citation: Ross BH, Lin Y, Corales EA, Burgos PV, Mardones GA (2014)
Structural and Functional Characterization of Cargo-Binding Sites on the µ4-Subunit of Adaptor Protein Complex 4.
PLoS ONE 9(2): e88147.

Published customer image:
Sheep anti Human TGN46 antibody used for the identification of the trans golgi network in MD-MB-231 cells by immunofluorescence.
Image caption:
APP redistributes from endosomes to the TGN upon overexpression of μ4-R283D-HA or μ4-R283D. MD-MB-231 cells were cotransfected with a plasmid encoding either of the indicated HA-epitope-tagged or untagged variants of μ4, and with a plasmid encoding APP-GFP carrying the double mutation F615P/D664A. After 24-h cells were fixed, permeabilized, stained for EEA1 and TGN46, and examined by fluorescence microscopy. Merging green, red, and blue channels generated the fourth image on each row; yellow indicates overlapping localization of the green and red channels, cyan indicates overlapping localization of the green and blue channels, magenta indicates overlapping localization of the red and blue channels, and white indicates overlapping localization of the red, green, and blue channels. Insets show 2× magnifications. Bar, 10 μm.

From: Citation: Ross BH, Lin Y, Corales EA, Burgos PV, Mardones GA (2014)
Structural and Functional Characterization of Cargo-Binding Sites on the µ4-Subunit of Adaptor Protein Complex 4.
PLoS ONE 9(2): e88147.

Published customer image:
Sheep anti Human TGN46 antibody used for the identification of the trans golgi network in H4 Neuroglioma cells by immunofluorescence.
Image caption:
APP redistributes from endosomes to the TGN upon overexpression of μ4-F255A. H4 neuroglioma cells were cotransfected with a plasmid encoding either of the indicated variants of μ4, and with a plasmid encoding APP-GFP carrying the double mutation F615P/D664A. After 36-h cells were fixed, permeabilized, stained for TGN46 and EEA1, and examined by fluorescence microscopy. Merging green, red, and blue channels generated the fourth image on each row; yellow indicates overlapping localization of the green and red channels, cyan indicates overlapping localization of the green and blue channels, magenta indicates overlapping localization of the red and blue channels, and white indicates overlapping localization of the red, green, and blue channels. Insets show 2× magnifications. Bar, 10 μm.

From: Citation: Ross BH, Lin Y, Corales EA, Burgos PV, Mardones GA (2014)
Structural and Functional Characterization of Cargo-Binding Sites on the µ4-Subunit of Adaptor Protein Complex 4.
PLoS ONE 9(2): e88147.

Published customer image:
Sheep anti Human TGN46 antibody used to visualize the cellular distribution of the trans-golgi network in HeLa cells by immunofluorescence.
Image caption:
Cellular distribution of tetherin. (A) Confocal analysis of HeLa cells showing the distribution of endogenous tetherin, detected with a specific antiserum. Cells that were fixed but not permeablized (left panel) allowed visualization of tetherin at the cell surface, while permeabilized cells revealed tetherin concentrated in a perinuclear compartment that was visible in ~50% of cells. This intracellular pool co-localized with a marker for the TGN (TGN-46), as shown by the PDM analysis in the upper right corner of the merged image, where positive co-localization is pseudocolored in orange. Scale bars represent 10 µM. (B) 293A cells were co-transfected with 10 µg HIV-1-pack and 100 ng of expression plasmids for either untagged tetherin or EGFP-tagged tetherin. Cell lysates were analyzed by Western blotting, using antibodies against GFP and tetherin. (C) Cell lysates and pelleted supernatant fractions (VLPs) from same experiment as (B) were probed for HIV-1 p24 expression. Both tetherin constructs inhibited VLP release. (D) HeLa and 293A cells were transfected with either 100 ng or 300 ng of the EGFP-tetherin plasmid. With 300 ng, a punctate pattern of EGFP fluorescence was observed throughout the cells; with 100 ng, the protein could only be detected using an anti-GFP antibody, that revealed an intense surface rim and a fainter PNC in both types of cells. Cells were fixed and permeabilized before staining. Scale bars represent 10 μM. (E) The intracellular concentration of EGFP-tetherin in transiently transfected HeLa cells (100 ng plasmid) was analyzed by confocal microscopy using anti-GFP antibody and specific markers for the TGN (TGN46) and recycling endosomes (endocytosed transferrin). The degree of co-localization was calculated using Pearson&s coefficients. Mean +/- SEM is shown for 20 individual cells analyzed.

From: Hauser H, Lopez LA, Yang SJ, Oldenburg JE, Exline CM, Guatelli JC, Cannon PM. HIV-1 Vpu and HIV-2 Env counteract BST-2/tetherin by sequestration in a perinuclear compartment.
Retrovirology. 2010 Jun 7;7:51.

Published customer image:
Sheep anti Human TGN46 antibody used to visualize the cellular distribution of the trans-golgi network in HeLa cells by immunofluorescence.
Image caption:
Redistribution of tetherin to an intracellular compartment by HIV anti-tetherin factors. (A) The percentage of HeLa cells displaying tetherin concentrated in a perinuclear compartment (PNC) was calculated for 100 cells, from either control (Ctrl.) cells or cells transfected with 2 µg of Vpu or ROD10 Env expression plasmids. Mean +/- SEM is shown for n = 2 independent experiments. (B) HeLa cells transfected with either Vpu (Vphu-HcRed) or ROD10 Env, showed increased concentration of tetherin in a perinuclear compartment (arrowed), that co-stained with the TGN marker, TGN46. The triple color merged image is shown. Scale bars represent 10 µM.

From: Hauser H, Lopez LA, Yang SJ, Oldenburg JE, Exline CM, Guatelli JC, Cannon PM. HIV-1 Vpu and HIV-2 Env counteract BST-2/tetherin by sequestration in a perinuclear compartment.
Retrovirology. 2010 Jun 7;7:51.

Published customer image:
Sheep anti Human TGN46 antibody used to visualize the cellular distribution of the trans-golgi network in HeLa cells by immunofluorescence.
Image caption:
Tetherin redistribution by HIV-1 and HIV-2 proviral clones. HeLa cells were either mock treated or transfected with 8 µg of HIV-1NL4-3 or HIV-2ROD10 proviral clones. Cells were fixed, permeabilized, and stained for endogenous tetherin (green), the TGN46 marker (blue), HIV-1 Vpu (red) or HIV-2 Env (red). Triple color merged images are shown. NL4-3 transfected cells showed tetherin co-localized with Vpu and the TGN. ROD10 transfected cells had two distinct appearances. ~25% of cells showed tetherin localized with a compact TGN marker (upper panels), while the majority of the cells had tetherin in a more diffuse perinuclear location that co-localized with more distorted TGN staining (lower panels). Scale bars represent 10 µM.

From: Hauser H, Lopez LA, Yang SJ, Oldenburg JE, Exline CM, Guatelli JC, Cannon PM. HIV-1 Vpu and HIV-2 Env counteract BST-2/tetherin by sequestration in a perinuclear compartment.
Retrovirology. 2010 Jun 7;7:51.

Published customer image:
Sheep anti Human TGN46 antibody used to visualize the cellular distribution of the trans-golgi network in HeLa cells by immunofluorescence.
Image caption:
Analysis of perinuclear concentration of tetherin by Vpu and ROD10 Env. (A) HeLa cells were incubated with anti-tetherin antibody at 4°C for 1 hour, followed by incubation at 37°C for either 15 or 45 minutes to allow endocytosis of antibody-tetherin complexes. The antibody was found in a perinuclear region by 15 minutes (arrowed), but became more diffuse by 45 minutes, suggesting that tetherin quickly exits this compartment and recycles back to the plasma membrane. Scale bars represent 10 µM. (B) HIV-1 VLPs produced in 293A cells in the presence of 100 ng of wild-type (WT) or YY-AA tetherin, with and without co-transfection of 2 µg Vpu (pCMV-Vphu). Expression of proteins was confirmed by Western blotting with specific antibodies; tetherin levels were visualized for both untreated and PNGase F treated cell lysates. Mean +/- SEM fold-enhancement of VLP release by Vpu is shown in presence of tetherin or tetherin (YY-AA), n = 2. (C) HeLa cells expressing EGFP-tetherin or EGFP-tetherin(YY-AA) were incubated for 1.5 hours, with or without 20 µg/ml cycloheximide, and subsequently fixed, permeabilized, and stained with anti-EGFP antibody. In the absence of drug, both WT and YY-AA EGFP-tetherin proteins were found at the cell surface and in a perinuclear region (arrowed). After cycloheximide treatment, only EGFP-tetherin produced this population, with EGFP-tetherin(YY-AA) forming a more punctuate pattern, dispersed throughout the cytoplasm. Two different cells are shown for the drug treatment. Scale bars represent 10 µM. (D) HeLa cells were co-transfected with 60 ng EGFP-tetherin(YY-AA) and 2 µg of expression plasmids for either Vphu-HcRed or ROD10 Env. Cells were incubated with or without cycloheximide, as above, and subsequently fixed, permeabilized, and stained with anti-EGFP (green), anti-TGN46 (blue) and anti-HIV-2 Env (red) antibodies. Vpu was detected by HcRed expression (red). EGFP-tetherin(YY-AA) was concentrated in a perinuclear compartment by Vpu or ROD10 Env, overlapping with the TGN marker, irrespective of cycloheximide treatment. Scale bars represent 10 µM.

From: Hauser H, Lopez LA, Yang SJ, Oldenburg JE, Exline CM, Guatelli JC, Cannon PM. HIV-1 Vpu and HIV-2 Env counteract BST-2/tetherin by sequestration in a perinuclear compartment.
Retrovirology. 2010 Jun 7;7:51.

Published customer image:
Sheep anti Human TGN46 antibody used to localize the trans-golgi network in HeLa cells by immunofluorescence.
Image caption:
C11ORF24 is localized on the trans-Golgi Network. (A) HeLa cells were transfected with a construct encoding human C11ORF24 tagged with GFP and fixed 18 hours later. Cells were co-stained with TGN46 (middle, red) and GM130 (right, blue). The second line represents a closer view of the Golgi apparatus outlined by a rectangle in the merge image. (B) HeLa cells were fixed and co-stained with TGN46, C11ORF24 and Giantin. Colocalization is very strong between TGN46 and C11ORF24 (column 4) whereas it is lower with Giantin (column 5). Line profiles of the C11ORF24 (green), TGN46 (red) and Giantin (blue) fluorescence intensities from the lines in the magnified views are shown in column 7. The colocalization between TGN46 and C11ORF24 is clearly visible throughout mitosis (lines 2-5). (C) HeLa cells were stained for C11ORF24 and imaged using dSTORM super-resolution microscopy. A Fire LUT was used to facilitate visualization. A magnified view of the boxed region of the left image is shown in the middle and a magnified view of the boxed region of the middle image is shown on the right. The cell contour is outlined in red.

From: Fraisier V, Kasri A, Miserey-Lenkei S, Sibarita J-B, Nair D, Mayeux A, et al. (2013) C11ORF24 Is a Novel Type I Membrane Protein That Cycles between the Golgi Apparatus and the Plasma Membrane in Rab6-Positive Vesicles.
PLoS ONE 8(12): e82223.

Published customer image:
Sheep anti Human TGN46 antibody used to localize the trans-golgi network in HeLa cells by immunofluorescence.
Image caption:
C11ORF24 is present on RAB6 positive transport carriers. (A) HeLa cells expressing GFP-C11ORF24 were imaged every second for 5 minutes by spinning disk microscopy. The red arrow indicates a small transport carrier whereas the green arrow point to a long tube connected to the Golgi apparatus. A magnified view of these structures is shown in line 2 and 3. (B) HeLa cells were fixed and co-stained with TGN46, C11ORF24 and GTP-RAB6. Colocalization is very strong between TGN46, C11ORF24 and GTP-RAB6. A line profile of the C11ORF24 (green), TGN46 (red) and GTP-RAB6 (blue) fluorescence intensities from the lines in the magnified views are shown in column 7. (C) HeLa cells expressing GFP-C11ORF24 and mCherry-RAB6 were imaged every second for 5 minutes by spinning disk microscopy. The arrow indicates a transport carrier positive for GFP-C11ORF24 (green) and for mCherry-RAB6 (red). A magnified view of this structure is shown lines 4-6. (D) Transport carriers were visualized using a kymograph that was made along the line drawn on the merge image. All the dynamic elements positive for GFP-C11ORF24 (left and merge green) were positive for mCherry-RAB6 (middle and merge red).

From: Fraisier V, Kasri A, Miserey-Lenkei S, Sibarita J-B, Nair D, Mayeux A, et al. (2013) C11ORF24 Is a Novel Type I Membrane Protein That Cycles between the Golgi Apparatus and the Plasma Membrane in Rab6-Positive Vesicles.
PLoS ONE 8(12): e82223.

Published customer image:
Sheep anti Human TGN46 antibody used to localize the trans-golgi network in HeLa cells by immunofluorescence.
Image caption:
C11ORF24 localization after Brefeldin A treatment. (A) HeLa cells were either untreated (Control), treated with Brefeldin A for 5 minutes or treated with Brefeldin A for 60 minutes. Cells were then fixed and stained with anti-C11ORF24 (green) and anti-TGN46 (red) antibodies. (B) HeLa cells were treated with cycloheximide and then either directly processed (monensin 0 min) or treated with monensin for 60 minutes. Cells were then fixed and stained with anti-C11ORF24 (green) and anti-GM130 (red) antibodies.

From: Fraisier V, Kasri A, Miserey-Lenkei S, Sibarita J-B, Nair D, Mayeux A, et al. (2013) C11ORF24 Is a Novel Type I Membrane Protein That Cycles between the Golgi Apparatus and the Plasma Membrane in Rab6-Positive Vesicles.
PLoS ONE 8(12): e82223.

Published customer image:
Sheep anti Human TGN46 antibody used for the localization of the trans-glogi network in COS-1 cells by immunofluorescence.
Image caption:
Co-localization analysis of SMAP2 with several RE/TGN proteins. COS-1 cells were fixed with TCA, permeabilized, and then stained for SMAP2 (green) and the indicated proteins (red). For evection-2, Myc-tagged evection-2 was transiently expressed and stained with anti-Myc antibody. For CTxB, cells were pulsed for 3 min with Alexa 594-CTxB, chased for 35 min at 37°C, and fixed. Fluorescence intensity profile along white dotted lines is shown in the right column. Blue lines in the graph indicate regions where SMAP2 do not co-localize with TfnR or TGN46. Pearson coefficient was obtained using multiple images (n > 12 cells). Data represent mean ± SD. Scale bars, 1 µm.

From: Matsudaira T, Uchida Y, Tanabe K, Kon S, Watanabe T, Taguchi T, et al. (2013) SMAP2 Regulates Retrograde Transport from Recycling Endosomes to the Golgi. PLoS ONE 8(7): e69145.

Published customer image:
Sheep anti Human TGN46 antibody used for the localization of the trans-golgi network in Vero cells by immunofluorescence.
Image caption:
APP relocalizes in infected cells. (A) APP (red) co-localizes with TGN46 (green) in a compact tuft to one side of the nucleus (DAPI, blue) representing the trans-Golgi network in mock infected cells. (B) In cells infected with VP26-GFP HSV1 (green), APP (red) is distributed in particles throughout the cytoplasm at 7 hr p.i. Nuclei are stained with DAPI (blue). (C) Western blotting of uninfected (u) and infected (i) cells with anti-APP, anti-actin (loading control) and anti-VP5 (viral capsid) demonstrates significant increased amound of APP in infected cells. Actin bands remain similar, and VP5, as expected, is only detected in lanes loaded with infected cell lysate. Note no new APP bands are detected in the infected versus uninfected cell lysates. (D) Isolated virus separated on 7.5% SDS-PAGE and stained with amido black for protein and probed for APP by Western blotting with the same antibodies used for immuno-fluorescence. Note that only the 90–110 kD doublet representing APP is detected by anti-APP, with no additional viral protein bands detected. See also Figure S2 for split channels and Figure S3 for histone staining.

From: Cheng S-B, Ferland P, Webster P, Bearer EL (2011) Herpes Simplex Virus Dances with Amyloid Precursor Protein while Exiting the Cell.
PLoS ONE 6(3): e17966.

Published customer image:
Sheep anti Human TGN46 antibody used for the immunolocalization of the trans-golgi network in HeLa cells by immunofluorescence.
Image caption:
Time course of ShigaB sulfation in annexin A1 or A2 depleted cells. (A) HeLa cells transfected with control or annexin A1 siRNA for 72 h were analyzed for the amount of ShigaB being sulfated at different time points after addition of StxB. Sulfation data are plotted as percentages of the amount of sulphated ShigaB after 30 min incubation with control siRNA treated cells. Black and white bars represent sulfation of ShigaB in control and annexin A1 siRNA treated cells, respectively. Data presented are the average of 3–4 independent experiments, each performed in parallel, error bars indicating standard error of the mean, *p<0.05; **p<0.005 indicates statistically significant change. (B) After the same treatment as in (A), cells were fixed and stained with antibodies against TGN46 and ShigaB. Top panel shows representative confocal pictures for 30 min incubation with StxB, scale bars 20 µm. Left graphic shows quantification of amount of ShigaB colocalized with TGN46. In the right graphic, mean intensity of ShigaB in the Golgi area for the same representative experiment is plotted as percentage of mean intensity in the whole cell. Data presented are the average of at least 35 cells per condition. Quantifications where obtained with ImageJ software, error bars indicating standard error of the mean.

From: Tcatchoff L, Andersson S, Utskarpen A, Klokk TI, Skånland SS, Pust S, et al. (2012) Annexin A1 and A2: Roles in Retrograde Trafficking of Shiga Toxin.
PLoS ONE 7(7): e40429.

Published customer image:
Sheep anti Human TGN46 antibody used for the immunolocalization of the trans-golgi network in HeLa cells by immunofluorescence.
Image caption:
Stx transport in annexin A1 depleted cells is regulated by PKCd and PLA2. (A) Golgi transport of Shiga toxin was evaluated as described in materials and methods by quantification of sulfated ShigaB in HeLa cells transfected with siRNA against annexin A1 or non targeting siRNA, pretreated with the indicated inhibitors. Data from Stx sulfation are plotted as percentages of the value obtained for HeLa cells transfected with control siRNA and treated with DMSO. The white and black bars represent ShigaB-sulf2 sulfation for control and annexin A1 knockdown cells respectively. Data presented are the average of 3 independent experiments, each performed in parallel, error bars indicating standard error of the mean. *p<0.05 indicates statistically significant change between annexin A1 knockdown cells and the corresponding control siRNA treated cells. (B) After treatment with either 5 µM ONO-RS-082 for 30 min or 30 µM MAFP for 1 h, HeLa cells were incubated for 30 min with ShigaB before fixation and staining as indicated in the materials and methods section with antibodies against TGN46 and ShigaB. Panel shows representative confocal pictures, scale bars 20 µm. Graphic shows quantification of amount of ShigaB colocalized with TGN46 in one representative experiment plotted as percentage of control condition. Data presented for one representative experiment (n?=?3) are the average of at least 30 cells per condition. Quantifications were obtained with Zen 2009 software from Zeiss, error bars indicating standard error of the mean.

From: Tcatchoff L, Andersson S, Utskarpen A, Klokk TI, Skånland SS, Pust S, et al. (2012) Annexin A1 and A2: Roles in Retrograde Trafficking of Shiga Toxin.
PLoS ONE 7(7): e40429.

Published customer image:
Sheep anti Human TGN46 antibody used for the immunolocalization of the trans-golgi network in HeLa cells by immunofluorescence.
Image caption:
Distribution of MPRs and TPST in annexin A1 depleted cells. (A) To visualize the localization of MPRs, cells were fixed, permeabilized and immunostained with a sheep anti-TGN antibody in combination with a mouse monoclonal anti-CD or -CI MPR antibody. In (B), cells were transfected with an EGFP-TPST2 expression plasmid 48 h after transfection with siRNA. TGN was stained as in (A). Scale bars 10 μm. The relative colocalization of MPR46, MPR300 or EGFP-TPST2 with TGN46 positive structures was quantified by ImageJ software. Graphs represent the average from 25 cells plotted as percentage of total fluorescence for each marker, for one representative experiment (n = 3), where error bars indicate standard deviation.

From: Tcatchoff L, Andersson S, Utskarpen A, Klokk TI, Skånland SS, Pust S, et al. (2012) Annexin A1 and A2: Roles in Retrograde Trafficking of Shiga Toxin.
PLoS ONE 7(7): e40429.