Fig 1: Selective cargo recycling takes place in enlarged Rab5-positive endosomes.HeLa cells, stably expressing mApple-Rab5 and transiently transfected with the cargos TfR-GFP (A,D,G), GFP-CDMPR (B,E,H) or GalT-GFP (C,F,I), were treated for 20 min with nigericin, washed and imaged over 3 hr, as described in Figure 4A. (A–C) Time-lapse images of representative endosomes to show cargo acquisition and removal relative to Rab5 recruitment. Time-lapse videos of the endosomes in (A,D,G) at 1 min interval are available in Figure 9—video 1, Figure 9—video 2, and Figure 9—video 3, respectively. (D–F) Corresponding graphs of MFI of Rab5 and specified cargo at the endosome in (A,B,C), respectively, over the time the endosome was detectable, to show prompt removal of TfR and CDMPR, and GalT remaining unchanged. (G–I) Averaged Rab5 and specified cargo acquisition and removal, representing 24, 24, and 17 endosomes, respectively. Error bars represent standard deviation. Three independent experiments were performed. Numerical data for all analyzed endosomes is available in Figure 9—source data 1, Figure 9—source data 2, and Figure 9—source data 3. Figure 9—source data 1.Quantification of acquisition and removal of TfR to and from endosomes in relation to Rab5. Figure 9—source data 2.Quantification of acquisition and removal of CDMPR to and from endosomes in relation to Rab5. Figure 9—source data 3.Quantification of acquisition and removal of GalT to and from endosomes in relation to Rab5.
Fig 2: Nigericin-induced enlarged compartments originate at the Golgi and contain trans-Golgi marker GalT, later found in ILVs, with most enlarged compartments resolved by 48 hr.Nigericin was added to HeLa cells for 20 min and washed away, and cells were processed for electron microscopy (A–C), imaged by time-lapse microscopy (D) or harvested for counting (E) at specified times after the wash. (A) Cells stained with osmium tetroxide and potassium hexacyanoferrate reveal large spherical compartments (cyan arrows) originating at the trans-face of the Golgi (magenta arrows) in nigericin-treated cells and, occasionally, in untreated cells. (B,C) Cells stably expressing GalT-GFP were stained with anti-GFP and 12 nM Gold-conjugated secondary antibody to reveal GalT-GFP at the Golgi (magenta arrows), the limiting membrane of the enlarged compartments (cyan arrows) as well as in ILVs of the enlarged MVBs at later time points (yellow arrows).
Fig 3: Short nigericin treatment leads to trans-Golgi vesiculation and subsequent acquisition of Rab5.(A) Images of untreated HeLa cells stably expressing mApple-Rab5 and transiently expressing GalT-GFP. Arrows point to puncta positive for both markers. (B–D) Nigericin was added to HeLa cells for 20 min and washed away, and cells were imaged by time-lapse microscopy (B), processed for electron microscopy (C) or for immunofluorescence (D) at specified times after the wash. (B,C) HeLa cells stably expressing GalT-GFP and transiently transfected with mApple-Rab5. (B) Representative kinetic of Golgi vesiculation post nigericin treatment as visualised with GalT-GFP. The selected vesiculated compartment (arrow), initially negative for Rab5 subsequently becomes positive for both markers. A time-lapse video of the endosome at 2 min interval is available in Figure 3—video 1. (C) Immuno-EM image of a cell at 2 hr post recovery, with 12 nm Gold-labelled GFP (green arrows) and 5 nm Gold-labelled mApple (red arrows) present at the enlarged compartments. Scale bar = 500 nm. (D) Images of cells stained with anti-GalT to reveal endogenous GalT presence at the enlarged compartments.
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