sheep antibody to tgn46  (Bio-Rad)

Journal: Frontiers in Molecular Biosciences

Article Title: Loss of ARF5 impairs recovery after lysosomal damage

doi: 10.3389/fmolb.2025.1699266

Figure Lengend Snippet: Lipid transfer proteins OSBP and ORP9 are recruited to damaged lysosomes in an ARF-independent manner. (A) HeLa cells were transfected with empty vector (shCTRL) or pLKO containing ARF1 or ARF5 shRNAs. Cells were treated or not with LLOME (0.5 mM) for 35 min, fixed and stained for endogenous OSBP (green), LAMP1 (red), and TGN46 (magenta), and imaged with confocal microscopy. Scale bars indicate 10 µm. (B) HEK293 cells stably expressing tagged TMEM192 were transfected with empty pLKO vector (shCTRL) or pLKO containing ARF1 or ARF5 shRNAs. Untreated and LLOME (1 mM)-treated cells were subjected to the LysoIP protocol. Precipitated lysosomes were immunoblotted for endogenous OSBP. (C) HeLa cells were transfected with empty vector (shCTRL), ARF5, or ARF6 shRNAs. Cells were treated or not with LLOME (0.5 mM) for 20 min, fixed and stained for ORP9 (green), LAMP1 (red), and F-actin (blue), and imaged with confocal microscopy. Scale bars indicate 10 µm. (D) Quantification of data in (C) n = 28 cells. Error bars represent mean +SD. Data were analyzed with two-way ANOVA with Dunnett’s multiple comparisons test with a single pooled variance. *p < 0.05.

Article Snippet: The antibodies we used were: rabbit antibody to LAMP1 (D2D11) (Cell Signaling Technology, Inc.), Cat No. 9091S, IF (1:400) WB (1:1200); mouse antibody to LAMP1 (1D4B) from Developmental Studies Hybridoma Bank; mouse antibody to GFP (Proteintech) Cat No.:66002-1-Ig WB (1:100,000); mouse antibody to HA (16B12) (Biolegend) WB (1:4000); rabbit antibody to mCherry (Sigma-Aldrich) Cat No.SAB2702295-100UL WB (1:10,000); rabbit antibody to OSBP (Sigma) Cat no. HPA039227 WB (1:1100) IF (1:100); rabbit antibody to ORP9 from Dr. Neale Ridgway, Dalhousie University, IF (1:1000); mouse antibody to Golgin97 (Molecular probes) Cat No. CDF4 A-21270 WB (1:500); rabbit antibody to ARF5 (Novus Biologicals) Cat No. NBP1-31005 WB (1:2500); sheep antibody to TGN46 (Serotec, Oxford United Kingdom); mouse antibody to Gal-3 (B-2) (Santa Cruz Biotechnology, Inc.) Cat No. sc-25279 IF (1:100); IRDye 800CW donkey anti-mouse secondary antibody (Li-COR) 926-32212 WB (1:10,000); IRDye 680RD goat anti-rabbit secondary antibody (Li-COR) 926-68071 WB (1:10,000); Alexa Fluor 488 donkey anti-mouse (ThermoFisher Scientific (Rockford, IL) IF (1:100); Alexa Fluor 488 donkey anti-rabbit (ThermoFisher Scientific (Rockford, IL) IF (1:100); Alexa Fluor 568 donkey anti-mouse (ThermoFisher Scientific (Rockford, IL) IF (1:100); Alexa Fluor 568 donkey anti-rabbit (ThermoFisher Scientific (Rockford, IL) IF (1:100).

Techniques: Transfection, Plasmid Preparation, Staining, Confocal Microscopy, Stable Transfection, Expressing

Journal: bioRxiv

Article Title: Mechanical forces stimulate Golgi export

doi: 10.1101/2025.09.02.673725

Figure Lengend Snippet: ( A ) Representative confocal microscopy images of fixed HeLa cells transfected with the GST-C1a-PKD DAG sensor, immunostained for TGN46, seeded over FN or PLL for the indicated times. Scale bars, 10µm. ( B ) SuperPlot (N∼10 cells per biological replicate; n=3 biological replicates) of the quantification of ( A ). A repeated-measures 2-way ANOVA test was performed. P values using Tukey’s post-hoc multiple comparison test are reported. ( C ) Representative images of HeLa cells transfected with the GST-C1a-PKD DAG sensor, immunostained with GM130, and seeded on FN-coated glass coverslips, stiff (30 kPa) or soft (2 kPa) PAA gels. ( D ) SuperPlot showing quantification of ( C ). ( E ) Representative confocal microscopy images of fixed HeLa cells transfected with the GST-C1a-PKD plasmid, immunostained with an α-TGN46, and subjected to a treatment with or without 10 µM Tubacin. Scale bars, 10 µm. ( F ) SuperPlot (N∼10 per biological replicate; n=3 biological replicates) showing quantification of ( E ). A two-sided parametric ratio paired t-test was used. The P value is indicated in the plot.

Article Snippet: Commercially available antibodies used were as follows: anti TGN46 (sheep) antibody was purchased from Bio-RAD (AHP500GT).

Techniques: Confocal Microscopy, Transfection, Comparison, Plasmid Preparation

Journal: bioRxiv

Article Title: Mechanical forces stimulate Golgi export

doi: 10.1101/2025.09.02.673725

Figure Lengend Snippet: ( A ) HeLa cells transfected with the PAUF-RFP plasmid were spread on FN for 4h and subjected to a treatment with (5 µM) or without CRT0066101, a PKD inhibitor, fixed, and imaged by confocal microscopy. Scale bar, 10 µm. (B) SuperPlot showing the number of CARTS per cell for each experimental condition. (C) Representative confocal microscopy images of fixed HeLa cells transfected with the GFP-mem plasmid, subjected or not to a treatment with CRT0066101. Scale bars, 10 µm; representative cell length scales shown. (D) SuperPlots showing individual cell measurements (N∼10 per biological replicate; n=3 biological replicates). A two-sided parametric ratio paired t-test was used. The P value is indicated in the plot. ( E ) Images of live RPE1-ManII-Halo stably expressing cells before and after treatment with 5 µM CRT0066101. The FLIM signal and lifetime are displayed. The Golgi apparatus was post-labelled using JF-646. Higher magnification is shown on the right. (F) SuperPlot showing FLIM values expressed as fluorescence lifetimes in ns was quantified for each experimental condition. (G) Representative confocal microscopy images of fixed HeLa cells (WT or TGN46 KO), transfected with the GFP-mem plasmid, and seeded over FN for the indicated times. Scale bars, 10µm. (H) SuperPlot showing individual cell measurements (N∼10 per biological replicate; n=3 biological replicates) quantifying adhesion area from images in ( G ). A repeated-measures 2-way ANOVA test was performed. P values using Tukey’s post-hoc multiple comparison test are reported. ( I ) Schematic representation of our findings (see text for details).

Article Snippet: Commercially available antibodies used were as follows: anti TGN46 (sheep) antibody was purchased from Bio-RAD (AHP500GT).

Techniques: Transfection, Plasmid Preparation, Confocal Microscopy, Stable Transfection, Expressing, Fluorescence, Comparison

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