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: The Journal of Cell Biology

Article Title: Multiple golgins are required to support extracellular matrix secretion, modification, and assembly

doi: 10.1083/jcb.202411167

Figure Lengend Snippet: Golgi organization is altered upon loss of GMAP210 or Golgin-160. (A and C) Maximum projection confocal images of WT and GMAP210 KO (A) and Golgin-160 KO (C) RPE1 cells immunolabeled for cis -Golgi (GM130, magenta), cis/medial -Golgi (giantin, green), and TGN (TGN46, blue) markers. Nuclei labeled with DAPI (grayscale). Scale bar, 10 µm. Inset scale bar, 1 µm. (B and D) Quantification of total giantin and TGN46 area and fragment number per cell from images represented in A and C. Individual dots represent one cell and are colored by replicate ( n = 3). Bars show the median from each replicate experiment. Statistical analysis was performed using a Shapiro–Wilk normality test and a Kruskal–Wallis significance test. (E–G) Tomographic reconstructions of Golgi structures in WT (E), GMAP210 KO (F), and Golgin-160 KO (G) cells. Segmented membranes are labeled as cisternae (blue/purple), dilated structures (red), tubulovesicular structures (yellow), and vesicles (green). (E ii, F ii, iii, and G ii iii) Single-slice images with segmentation. (F ii and iii) Open arrows point to invaginations within spherical regions of tubulovesicular structures. (G ii and iii) Double-headed arrows indicate top-to-bottom fenestrations in cisternae, closed-headed arrows indicate budding structures at the nuclear envelope, and pink arrow shows frustrated budding/fusion intermediate. (E i, iii, iv, F i, iv, v, and G i, iv, v) 3D rendering of segmentation. (E–G i) Scale bar, 1 µm.

Article Snippet: TGN46 RRID:AB_324049 , Bio-Rad , AHP500 , 170720 , Full-length human protein , IF 1:1,000 , PFA/MeOH.

Techniques: Immunolabeling, Labeling

Journal: The Journal of Cell Biology

Article Title: Multiple golgins are required to support extracellular matrix secretion, modification, and assembly

doi: 10.1083/jcb.202411167

Figure Lengend Snippet: BGN-SBP-mSc localization in mutant cells. (A) Widefield maximum projections of cells stably expressing BGN-SBP-mSc (blue) and immunolabeled for the ERGIC (ERGIC53, green), cis/medial -Golgi (giantin, magenta), and TGN (TGN46, grayscale). Scale bar in main image, 10 µm; and insert, 1 µm. (B and C) Line-scan fluorescence intensity readings across Golgi elements measured at key time points in the RUSH assays shown in and ; and , , , , , and . The region measured is shown in inset. Line traces show (B) BGN-SBP-mSc (magenta trace) or (C) SPARC-SBP-mSc accumulates adjacent to ManII-positive Golgi elements (green trace, middle column) and then colocalizes with ManII-BFP (right column). Fluorescence measurements are normalized to the maximal point of each trace to account for differential expression and photobleaching rates of each fluorescent protein. Arrows highlight peak for each protein. ManII, mannosidase II.

Article Snippet: TGN46 RRID:AB_324049 , Bio-Rad , AHP500 , 170720 , Full-length human protein , IF 1:1,000 , PFA/MeOH.

Techniques: Mutagenesis, Stable Transfection, Expressing, Immunolabeling, Fluorescence, Quantitative Proteomics