Gfp booster atto488

For immunofluorescence analysis we used the following antibodies: sheep anti-Cnn (1:500) (Cottee et al., 2013 (link)); rabbit anti-PLP (PPHA) (1:500) (Martinez-Campos et al., 2004 (link)); mouse anti-TACC (1:500) (Gergely et al., 2000 (link)); rat anti-Asl (1:500) and rat anti-Spd2 (1:500) (Baumbach et al., 2015 (link)). Secondary antibodies were from Molecular Probes (Life Technologies): Alexa Fluor 405, 488 and 592 (all used at 1:1000). To enhance GFP fluorescence for 3D-SIM on fixed embryos we used GFP-Booster ATTO488 1:500 (Chromotek). For western blotting rabbit anti-PLP (PPHA) (1:1000) (Martinez-Campos et al., 2004 (link)) and mouse anti-actin (1:1000) (Sigma) were used. Secondary antibodies conjugated to IRDye 680 and IRDye800 were used at a concentration of 1:10,000 and blots imaged on an Odyssey CLx imager (LI-COR).

Hela or HeLa-FRT UBAP2L KO or parental cells were seeded in six-well dishes with coverslips at 25% confluency. Cells were transfected the day after with 250 ng DNA and 1 μL or jet OPTIMUS (Polyplus) reagent overnight in DMEM media supplemented with FBS (10%) (HyClone) and PenStrep (1%) (Thermo Scientific). Media was changed to media containing 0.5 μM sodium arsenite for 30 min to induce stress granules formation. After washing with PBS cells were fixed for 20 min with 4% formaldehyde in PBS. Cells were premeballized for 10 min with PBS 0.5% Triton-100. Following three 5-min washes with PBS-T (0.05% Tween), 25 mM Glycine was incubated overnight at 4 °C. Coverslips were blocked in TBST (0.05% Tween) 3% BSA for 45 min at room temperature. Primary antibodies (anti-G3BP1 mouse abcam #ab56574, anti-FXR1 mouse clone 6BG10 Milipore #05-1529, GFP booster atto488 (Chromotek)) were incubated at 1:400 dilution in 3% BSA TBST (0.05% Tween) overnight at 4 °C. Following three 5-min washes with TBST (0,05% Tween), coverslips were incubated with secondary antibodies for 1 h at room temperature. Coverslips were mounted in MOWIOL mounting solution (Calbiochem #475904) and imaged on a Delta-Vision Elite microscope (DeltaVision) with 60x oil objective. Data were analyzed in Fiji and plotted with a Prism 9 GraphPad software.

Primary antibodies anti-EEA1 (ab70521), anti-58K Golgi (ab27043), antipericentrin (ab28144), and anticatalase (ab110292) were purchased from Abcam, anti-TGN46-8 (SAB4200355) from Sigma and anti-Tom20 (sc-17764) from Santa Cruz Biotechnology. All primary antibodies were monoclonal mouse antibodies. Polyclonal secondary antimouse antibodies conjugated to Atto647N (50185, Sigma Aldrich) as well as isotype-specific secondary antimouse antibodies conjugated to Atto647N (610-156-041, Rockland Immunochemicals) and Alexa Fluor 568 (A-21124, Thermo Fisher Scientific) were used for detection. The nanoboosters—single-domain alpaca antibody fragments covalently coupled to fluorescent dyes—GFP-Booster Atto488 and RFP-Booster Atto594 (Chromotek) were used to boost eYFP and mCherry fluorescence, respectively. Live cell stains SiR-tubulin, SiR-actin, and SiR-lysosome were from Spirochrome and MitoTracker Deep Red FM was from Thermo Fisher Scientific.

As primary antibodies we used the HPV16-L1 detecting antibody 16L1-312F (mouse monoclonal, diluted 1:200; [29 (link)]) and a rabbit polyclonal antibody raised against the V5-tag (diluted 1:5000; cat# ab9116, Abcam, Cambridge, UK). In addition, we employed GFP-Booster Atto488 (Chromotek, cat# gba488, Planegg-Martinsried, Germany) and RFP-Booster Atto594 (cat# rba594, Chromotek, Planegg-Martinsried, Germany), both diluted 1:200. As secondary antibodies we used AlexaFluor488 labelled donkey anti-mouse (cat# A-21202, Carlsbad, CA) and AlexaFluor594 labelled donkey anti-rabbit (cat# ab150064, Abcam). For transfection, we used the previously described plasmids encoding CD63-GFP [18 (link)], V5-OBSL1 [27 (link), 30 (link)] and CD151-RFP [31 (link)].