Duolink in situ detection reagents red

Cells grown on coverslips were pre-extracted in 0.5% NP40 on ice for 4min then washed once with PBS an fixed with 4% formaldehyde in PBS for 15 min at room temperature, washed three times with PBS, blocked with 3% BSA/10% fetal bovine serum and incubated with antibodies mouse RNAPII 8WG16Pol 1:200 and rabbit PCNA 1:200 overnight at 4°C. PLA was performed following the manufacturer’s instructions using the Duolink anti-Mouse MINUS and anti-Rabbit PLUS In Situ PLA probes and the Duolink In Situ Detection Reagents Red (Olink Bioscience). Images were acquired using a Zeiss AxioImager M1, equipped with a Hamamatsu digital camera and the Volocity software (Perkin Elmer). PLA foci were analyzed using ImageJ (https://imagej.nih.gov/ij/).

Cells were plated on coverslips at density 5 × 10⁴ in 24-well plates and left in culture conditions overnight. The next day cells were pre-extracted in CSK buffer (10 mM PIPES [pH 6.8], 100 mM NaCl, 300 mM sucrose, 3 mM magnesium chloride, 1 mM EGTA, and 0.5% Triton X-100) fixed with 5% formaldehyde (Thermo Scientific) for 10 min, permeabilized with PBS containing 0.5% (v/v) NP-40 for 5 min, and blocked for 30 min with goat serum (5%) in PBS. PLA was performed following the manufacturer’s instructions using the Duolink anti-Mouse MINUS and anti-Rabbit PLUS In Situ PLA probes and the Duolink In Situ Detection Reagents Red (Olink Bioscience). Images were acquired with a Zeiss Axio Imager M1 microscope equipped with an ORCA-ER camera (Hamamatsu) and analyzed using Volocity 6.3 software (Improvision).

Ubiquitination of GFP-tagged TaMAB2 in transgenic protoplasts was tested using a Duolink In Situ proximity ligation assay (PLA) assay (OLINK Bioscience, Uppsala, Sweden). The primary antibodies were mouse monoclonal anti-GFP (1:400, 11814460001, Roche) and rabbit polyclonal anti-ubiquitin antibody (1:2,000, AB1690, Chemicon International). Negative controls were performed using A. thaliana Col-0 wild type protoplasts, in which the anti-GFP antibody should have no targets.
Protoplasts were isolated from 2-week old seedlings (Zhai et al., 2009 (link)) of A. thaliana overexpressing TaMAB2-GFP (lines 80 and 82) in Col-0 genetic background. Protoplasts were fixed with 4% paraformaldehyde and adhered to positively charged silane-coated slides. After rehydration in phosphate-buffered saline, protoplasts were blocked for 30 min using Duolink PLA Blocking Solution and incubated in Duolink PLA Antibody Diluent (OLINK Bioscience) containing primary antibodies. Primary antibody incubation lasted for 2 h at room temperature followed by overnight incubation at 4°C. Ubiquitination of GFP-tagged protein was detected using Duolink In Situ PLA probes and Duolink In Situ Detection Reagents Red according to manufacturer’s instructions (OLINK Bioscience). A minimum of 30 protoplasts emitting a PLA signal was analyzed for both lines in three biological replicates.

The PLA assay was performed using the Duolink in situ reagents (Olink Biosciences) in according with the manufacturer’s specifications. The cells were fixed with 4% PFA in PBS for 15 min at room temperature. After blocking with 0.1% Triton X-100 and 0.5% BSA dissolved in PBS, the cells were incubated overnight at 4 °C with primary antibodies. Following incubation with Duolink PLA anti-rabbit Plus and anti-mouse Minus probes (1:5 dilution, Olink Bioscience) at 37 °C for 1 h, ligation, rolling circle amplification, and detection were performed using the Duolink In Situ Detection Reagents Red (Olink Bioscience). Nuclei were counterstained with DAPI. PLA signals were documented by a Leica SP8 confocal microscope and quantified by using cell image analysis software CellProfiler^{63 (link)}.