Medical x ray blue mxbe film

SDS-PAGE was performed (150 V, RT) and proteins were transferred to polyvinylidene difluoride membranes (200 mA). The membranes were blocked (1 h, RT), incubated with primary antibody (nectin-3: 1:1000, catalog no. ab63931, Abcam; β-actin: 1:5000, catalog no. A1978, Sigma) overnight (4 °C), washed 3 × in Tris-buffered saline/0.1% Tween-20 (TBS-T; 10 min, shaking) and incubated with secondary HRP-conjugated antibodies (in blocking buffer, 1 h, RT; anti-rabbit P-100, 1:5000; anti-mouse P-200, 1:5000; Vector Laboratories). After TBS-T washing, the signal was developed (ECL Western Blotting Detection Reagent; GE Healthcare) and detected on Medical X-ray Blue/MXBE Film (Carestream) using a Fuji Film Processor (FPM-800A). The optical density of the nectin-3/actin bands was quantified (GeneTool program).

The procedure was performed as described in Mazur et al. (2021) (link). In brief, for immunoprecipitation of GFP-fused proteins, 400 µg of crude protein extract from seedlings treated with H₂O₂ or MV was incubated with 10 µL of GFP-Trap^®_A (Chromotek) for 2.5 h with gentle rocking. After intensive washing, agarose beads with bound immunocomplexes were suspended in 20 mM Tris–HCl, pH 7.5 supplemented with 150 mM NaCl and 4 µg of Myelin Basic Protein (Sigma-Aldrich) per sample. To each sample, ATP supplemented with 1 μCi of [γ³²P]ATP in kinase buffer (25 mM Tris-HCl, pH 7.5, 5 mM EGTA, 1 mM DTT, 30 mM MgCl₂) was added to 50 μM final concentration. After 15 min of incubation at 37°C samples were mixed with Laemmli sample buffer and incubated for 3 min at 95°C with vigorous shaking. Proteins were separated on 12% SDS polyacrylamide gel and signal was detected on Medical X-ray Blue/MXBE Film (Carestream).

Standard nucleoside phosphoramidites and 5′-dimethoxytrityl-2′-propargyluridine 3′-O-(N,N-diisopropyl-2-cyanoethyl) phosphoramidite were purchased from ChemGenes Corporation (Wilmington, MA, USA). [(3,3′-Iron-1,2,1′,2′-dicarbollide)⁻¹]ate (-1) cesium salt (ferra(III) bis(dicarbollide), ([C₂B₉H₁₁)²]⁻), FESAN), was purchased from Katchem (Režn/Prague, Czech Republic). Radiolabeled adenosine 5′-triphosphate ([γ-³²P]-ATP) was purchased from Hartmann-Analytik (Braunschweig, Germany), and T4 polynucleotide kinase and kinase buffer were purchased from BioLabs (Ipswich, MA, USA). Autoradiography double-coated films Medical X-ray blue (MXBE film) were purchased from Carestream (New York, NY, USA). The developer reagent and fixer reagent were purchased from Kodak Processing Chemicals (Sigma Aldrich, St. Louis, MO, USA). RNase H (Ribonuclease H, E. coli), an RNA-specific endonuclease that cleaves the RNA within RNA/DNA hybrids was purchased from EURx, (Gdańsk, Poland).