Molecular imager pharosfx system

ADP-ribosylation of Cy3-labelled DNA and RNA oligonucleotides was performed as described earlier (101 (link),130 (link)). Proteins for the assay were purified as described in the same studies. Briefly, 10 μL reactions were prepared in ADP-ribosylation buffer (20 mM HEPES–KOH (pH 7.6), 5 mM MgCl₂ and 1 mM DTT). The reactions contained 1 μM Cy3-labelled RNA or DNA oligonucleotide, 3 μM PARPs, PARP10 ART (residues 868–1025), or PARP14 WWE-ART (residues 1459–1801), and 500 μM NAD⁺. The reactions were incubated for 1 h at 37°C and stopped by adding 50 ng/μl Proteinase K and 0.15% SDS followed by incubating at 50°C for 30 min. Finally, the reactions were mixed with 2× TBE urea sample buffer (8 M urea, 20 μM EDTA (pH 8.0), 20 μM Tris–HCl (pH 7.5), and bromophenol blue) and loaded on a pre-run 15% denaturing urea polyacrylamide gel electrophoresis (PAGE) gel. The gels were run at 7 W/gel and imaged using the Molecular Imager PharosFX system (BioRad) with laser excitation for Cy3 at 532 nm.

NCS enzyme assays were performed as described previously8 (link). Briefly, reaction mixtures containing recombinant protein, 1 nmol [8-¹⁴C]-dopamine and 10 nmol 4-HPAA were incubated for 1.5 h at 37 °C. Reactions were subsequently spotted onto silica gel 60 F₂₅₄ TLC plates and developed in n-butanol:acetic acid:water (4:1:5, v/v/v). Autoradiogram visualization and analysis was performed using a Molecular Imager PharosFX system and Quantity One software (Bio-Rad).

The gel images from both the LF and HF groups were scanned on a Molecular Imager Pharos FX system (Bio-Rad, Hercules, CA, USA). Image analysis including image editing, spot finding, quantitation, and matching, was carried out using Quantity One Software (Bio-Rad) and PD Quest version 8.0 2-D gel analysis software (Bio-Rad, Hercules, CA, USA). The protein spots were detected by following the PD Quest software instructions using the following parameters: horizontal and vertical streaking removal with a radius of 33, smoothing by Power Mean filter with kernel size 3 × 3, speckle removal at a sensitivity of 50. The densities of protein spots were normalized using the Local Regression Model. The spots were then quantitatively compared between LF and HF groups using the approach by Sun et al.^{43 (link)}. Protein spots were considered to be differentially expressed if the difference between the average of spot densities between the two groups was twofold greater or lesser.

The DTX3L RD or DTX3L full length proteins at varying concentrations were incubated in 10-μl reactions with 0.5 μM E35_5′P_RNA_3Cy3 in the buffer containing 20 mM HEPES (pH 7.5), 50 mM KCl, 5 mM MgCl₂ and 1 mM DTT. After incubation on ice for 0.5 h, the reaction mixtures were separated on a pre-run 6% native PAGE gel and then the gel was imaged using the Molecular Imager PharosFX system (BioRad) with laser excitation for Cy3 at 532 nm.

The inoculated plants were maintained in a growth chamber at 25 °C with 16 h light/8 h dark periods. After 3 days post inoculation (dpi), the control plants were monitored regularly under UV light (Blak-Ray B-100 AP Lamp, Analytik Jena, Jena, Germany) to visualize the silencing phenotype. At 6 dpi all the plants were recorded using a camera (Canon EOS700D, 18–55 mm Lens, Canon, Mannheim, Germany) under UV light and leaves were scanned using a laser scanner (Molecular Imager^® PharosFX™ Systems, BioRad, Hercules, CA, USA) at 50 µm/pixel, in GFP (excitation at 488 nm, emission at 530 nm) and chlorophyll (excitation at 488 nm, emission at 695 nm) channels.
The GFP and the chlorophyll images were merged and the GFP expression was quantified in the limited area of infiltrated patches. GFP expression was measured by FIJI image analysis in treated, non-necrotic areas, and the acquired values were normalized using non-treated areas. The final values were plotted in bar charts and mean value graphs together with the actual data points.