sheep polyclonal anti human tgn46  (Bio-Rad)

Journal: EMBO Reports

Article Title: Cargoes move from cis to trans -Golgi compartments and concentrate in the TGN before exiting

doi: 10.1038/s44319-025-00548-9

Figure Lengend Snippet: ( A ) The individual traces of the iFRAP data presented in Fig. are shown. ( B ) Representative traces from A were normalized in two different ways. As presented in ( A ) or they were normalized by total fluorescence to account for bleaching (orange). The curves were qualitatively similar with flat phase followed by an exponential decrease. ( C ) The intensity levels of background (BG; area with no cells), ER and Golgi were measured before and after bleaching in the iFRAP protocol. The bleaching was efficient in the ER while there was little to no change in the Golgi fluorescence (mean ± SD; n > 3). ( D ) VSVG-GFP-RUSH construct localization with Golgi markers (GM130 and TGN46) from the replicate 2 of Fig. . The positions of VSVG-GFP-RUSH and Golgi marker peaks were normalized, setting GM130 as 0 and TGN46 as 1. Measurements obtained using line scan analysis (red) are compared with those from the GLIM based automated method (blue) within the same experiment. Each data point represents an individual measurement, with results from both methods shown side-by-side for direct comparison. The relative position of the VSVG-GFP-RUSH construct is plotted at each indicated time point (median ± SE; n is indicated in the figure, n.s = not significant; [Student’s t test]). .

Article Snippet: Polyclonal Sheep anti-human TGN46 , BioRad/AbD-Serotec , Cat #AHP500G RRID:AB_323104.

Techniques: Fluorescence, Construct, Marker, Comparison

Journal: EMBO Reports

Article Title: Cargoes move from cis to trans -Golgi compartments and concentrate in the TGN before exiting

doi: 10.1038/s44319-025-00548-9

Figure Lengend Snippet: ( A ) HeLa cells were transfected with VSVG-GFP-RUSH constructs overnight and then treated with nocodazole (33 μM) for 3 h at 37 °C. Biotin was then added to cells and at indicated times the cells were fixed and stained for GM130 and TGN46 to mark the cis - and trans -Golgi, respectively. The relative position of the cargo with respect to GM130 and TGN46 is shown. Bar: 1 μm. ( B ) The position of the cargo and Golgi marker peaks were normalized considering the position of the GM130 peak to be 0 and TGN46 to be 1. The relative positions of cargo peaks over time are plotted (mean +/− SE; Data points ( n > 70) from three independent experiments are represented by grouped circles in three different shades of the same color). ( C ). HeLa cells were transfected with E-cadherin-GFP-RUSH construct overnight and then treated with nocodazole (33 μM) for 3 h at 37 °C. Biotin was then added to cells and at indicated times the cells were fixed and stained for GM130 and TGN46 to mark the cis - and trans -Golgi, respectively. The position of the cargo and Golgi marker peaks were normalized considering the position of the GM130 peak to be 0 and TGN46 to be 1. The relative positions of cargo peaks over time are plotted (mean +/− SE; Data points ( n > 70) from three independent experiments are represented by grouped circles in three different shades of the same color). ( D ) HeLa cells were co-transfected with VSVG-Halotag and GalT-CFP, and the cargo was accumulated in the ER at 40 °C overnight. Cells were treated with nocodazole (33 μM) for 3 h, then shifted to 32 °C to allow the cargoes to enter the Golgi, and fixed at each indicated time. Cells were then stained for GM130 and TGN46 to mark the cis - and trans -Golgi, respectively. The position of the cargo and Golgi marker peaks were normalized considering the position of GM130 peak to be 0 and TGN46 to be 1. The relative positions of cargo peaks over time are plotted (mean +/− SE; Data points ( n > 70) from three independent experiments are represented by grouped circles in three different shades of the same color. n.s = not significant (*** P = 1.96e-33; 3.2e-07; 6.89161e-07 [Student’s t test]). .

Article Snippet: Polyclonal Sheep anti-human TGN46 , BioRad/AbD-Serotec , Cat #AHP500G RRID:AB_323104.

Techniques: Transfection, Construct, Staining, Marker

Journal: EMBO Reports

Article Title: Cargoes move from cis to trans -Golgi compartments and concentrate in the TGN before exiting

doi: 10.1038/s44319-025-00548-9

Figure Lengend Snippet: ( A ) HeLa cells were transfected with VSVG-GFP and kept overnight at 40 °C. Then cells were treated with nocodazole for the indicated amount of time and shifted to 32 °C for 25 min and then fixed and prepared for immunofluorescence with the indicated markers. White boxes represent insets. Bar: 10 μm. ( B ) Quantification from ( A ) of the number of ministacks showing the indicated markers. Mean +/− SD. Data points ( n > 290) from 3 independent experiments. ( C ) The Pie chart shows the percentage of VSVG-mCherry-containing tubules exiting from TGN and those whose origin is unclear ( n = 13). We have not observed any tubule originating from cis -Golgi. ( D HeLa cells were transfected with VSVG-mCherry-RUSH construct overnight and then biotin was added for 1 h. The cells were fixed and stained for GM130 and TGN46 to mark the cis -Golgi and TGN respectively. White boxes represent insets. Bar: 5 μm. .

Article Snippet: Polyclonal Sheep anti-human TGN46 , BioRad/AbD-Serotec , Cat #AHP500G RRID:AB_323104.

Techniques: Transfection, Immunofluorescence, Construct, Staining

Journal: EMBO Reports

Article Title: Cargoes move from cis to trans -Golgi compartments and concentrate in the TGN before exiting

doi: 10.1038/s44319-025-00548-9

Figure Lengend Snippet: ( A ) HeLa cells stably transfected with hGH-FM-GFP were treated with nocodazole (33 μM) for 3 h at 37 °C. Then they were incubated with DD solubilizer for the indicated times at 37 °C. The cells were fixed and stained for GM130 and TGN46 to mark the cis - and trans -Golgi, respectively. The position of GFP-FM4-hGH relative to GM130 and TGN46 is shown. Bar: 1 μm. ( B ) The position of the GFP-FM4-hGH and Golgi marker peaks was normalized, considering the position of the GM130 peak to be 0 and TGN46 to be 1. The relative position of GFP-FM4-hGH peaks over time is plotted (mean +/− SE; Data points ( n > 70) from three independent experiments are represented by grouped circles in three different shades of the same color). ( C ) Cells prepared as in ( A ) were fixed 20 min after the addition of DD solubilizer and prepared for EM. The sections were labeled with anti-GFP antibody (10 nm gold). ( D ) HeLa cells stably transfected with GFP-FM4-hGH were incubated with DD solubilizer for indicated times at 37 °C. The culture supernatant and cell lysate were collected and analyzed by Western blotting with anti-GFP antibody. While the intracellular version of hGH-FM-GFP was approximately at 75 kDa, the secreted form of the protein was consistently at ~55 kDa. Arrowhead indicates full-length form of GFP-FM4-hGH and double arrowhead indicates furin cleaved form. Asterisk indicates a likely non-specifically cleaved protein since it is not secreted. .

Article Snippet: Polyclonal Sheep anti-human TGN46 , BioRad/AbD-Serotec , Cat #AHP500G RRID:AB_323104.

Techniques: Stable Transfection, Transfection, Incubation, Staining, Marker, Labeling, Western Blot

Journal: EMBO Reports

Article Title: Cargoes move from cis to trans -Golgi compartments and concentrate in the TGN before exiting

doi: 10.1038/s44319-025-00548-9

Figure Lengend Snippet: ( A ) HeLa Cells were transfected with VSVG-GFP and kept overnight at 40 °C. Then cells were shifted to 32 °C for 1 h and then fixed and prepared for EM. The sections were labeled for GFP (15 nm gold) and TGN46 (10 nm gold; indicated by blue circles). Bar: 100 nm. ( B ) HeLa Cells were transfected with VSVG-GFP-RUSH, GPI-GFP-RUSH or LAMP1-GFP-RUSH constructs and kept at 37 °C. Then cells were incubated with 1 μM biotin for 60 min at 37 °C. The cells were fixed and stained for GM130 and TGN46 to mark the cis - and trans -Golgi, respectively. The relative position of the cargo relative to the GM130 and TGN46 is shown. Bar: 2 μm. ( C ). In HeLa cells treated as in ( B ), the position of each indicated RUSH construct and Golgi marker peaks were normalized, considering the position of the GM130 peak to be 0 and TGN46 to be 1. The relative position of each indicated RUSH construct peak at 60 min is plotted (mean +/− SE; Data points ( n > 70) from three independent experiments are represented by grouped circles in three different shades of the same color). ( D ) HeLa cells were transfected with E-cadherin-GFP-RUSH constructs overnight and then treated with nocodazole (33 μM) for 3 h at 37 °C. The cells were treated with cycloheximide for the last 1 h at 37 °C. Biotin was then added to cells to allow the cargoes to enter the Golgi ministacks for 60 min. Then the fluorescence in the cell, except for a few Golgi ministacks, was photobleached (iFRAP). Left panel: the exit of the cargoes from the Golgi ministack was then monitored, and the fluorescence in the Golgi area was calculated over time and represented as normalized to the initial (after iFRAP) fluorescence levels (mean +/− SE; n = 2; 5 ministacks). Right panel: the data for the E-cadherin-GFP-RUSH exiting from Golgi ministacks fit well with a monoexponential decay (red) with the following rate constants: 0.005839 min -1 , with an R-squared value > 0.97. ( E ) Schematic representation of ( C ) of the iFRAP protocol for E-cadherin in cells treated with nocodazole. .

Article Snippet: Polyclonal Sheep anti-human TGN46 , BioRad/AbD-Serotec , Cat #AHP500G RRID:AB_323104.

Techniques: Transfection, Labeling, Construct, Incubation, Staining, Marker, Fluorescence

Journal: Nature

Article Title: A coronavirus assembly inhibitor that targets the viral membrane protein

doi: 10.1038/s41586-025-08773-x

Figure Lengend Snippet: a , Setup of the time-of-drug-addition assay (TOA). b , c , Ten hr p.i. intracellular vRNA b , and infectious viral particles c , were quantified (mean ± s.d., n = 3 biologically independent experiments, one-way ANOVA followed by Dunnett’s multiple comparisons with the DMSO control). d , TOA assay using VeroE6-H2B-mCherry cells and rSARS-CoV-2-mNeonGreen. e , Quantification of %infected cells (mNeonGreen + and mCherry + VeroE6 cells) relative to the DMSO control (mean ± s.d., n = 6 biologically independent experiments). f , Virus kinetics experiment with the same setup (compounds added at 0 h p.i.) and quantification of infected cells at different time points. In a-f , hydroxychloroquine was tested at 10 µM, CIM-834 and nirmatrelvir at 1 µM. g , CIM-834 does not influence the co-localization of M and TGN46, a marker for the trans-Golgi network. Data (mean ± s.d.) from two independent experiments. h , TEM (top) and tomographic reconstruction (bottom) of cytoplasmic regions of DMSO- or CIM-834(1 µM)-treated SARS-CoV-2 infected cells (10 h p.i.). a1, a2, Details of boxed regions highlighted in Fig. , showing additional examples of assembly sites located both at the center of the DMV cluster and at its periphery, in proximity of the terminal region of Golgi cisternae (G). m, mitochondrion. b1, b2: Magnified regions from Fig. , showing concentric stacks of membranes (orange dashed lines) detected in CIM-834 treated cells and their proximity to DMVs (*). c1,c2: Slices from tomographic acquisition of DMSO (c1) and CIM-834-treated cells (c2), highlighting the regions magnified respectively below (d, f) or in Fig. (c, e). Section thickness: 70 nm ( a1,a2,b1,b2 ). Tomographic section thickness: 0.78 nm ( c1,c2 ). i , Inhibition of the activities of purified, recombinant enzymes assayed in vitro in the presence of CIM-834 and appropriate reference compounds.

Article Snippet: The trans-Golgi network was visualized using polyclonal sheep anti-human TGN46 antibodies (AHP500, BioRad,1:500), followed by incubation with Cy3-conjugated donkey anti-sheep IgG antibodies (Jackson ImmunoResearch, 713-165-147, 1:500).

Techniques: Control, Infection, Virus, Marker, Inhibition, Purification, Recombinant, In Vitro