Luminometer plate

Protein synthesis inhibition activity for non-reduced or reduced S. nigra RIPs (SNA-I, SNA-V or SNLRP) was determined using the TnT T7 Quick Coupled Transcription/Translation System Kit (Promega, Mannheim, Germany) based on a cell-free system [20 (link)]. The lectins SNA-II and SNA-IV were also included in this assay. According to manufacturer’s instructions, the prepared mixture was incubated at 30°C for 10 min and chilled on ice. Afterwards, 2 μl PBS or PBS containing different concentrations of S. nigra RIPs or lectins were added to the reaction mixture and incubated for 30 min at 30°C. After addition of 35 μl nuclease-free water at room temperature the reaction samples were transferred to a luminometer plate (Greiner Labortechnik, Frickenhausen, Germany) containing 5 μl luciferase assay reagent at 25°C. The relative luciferase activities of the samples were determined at 562 nm for 10 sec using a microtiter top plate reader (Infinite 200, Tecan, Mannedorf, Switzerland) with an initial delay of 2 sec.

To study translation inhibition in cellula, a total of 3,000 HeLa cells (HG1-luc2-IRES-tCD cells [20 (link)]) were seeded per well in a 96-well plate (Greiner) and incubated at 37°C and 5% CO₂ for 24 h. After replacing the medium, the cells were exposed to medium supplemented with 100 nM S. nigra RIPs and SNA-II, and incubated at 37°C and 5% CO₂ for another 24 h. Cycloheximide (10 μg/ml, Sigma-Aldrich) was used as the control treatment [33 (link)]. Luciferase activity in cell extracts was assessed using the Promega Luciferase Assay System according to the manufacturer’s instructions (Promega, Mannheim, Germany). After washing with PBS, cells were lysed with lysis buffer (Promega luciferase kit) for 2 min and half of the cell lysate was transferred to the luminometer plate (Greiner). After addition of the D-Luciferin substrate, luminescence was measured for a period of 10 seconds. Recorded signals were normalized to the amount of viable cells as measured in a subsequent Presto blue assay. Four technical replicates were performed for each protein concentration, and each experiment was repeated three times.

The protein synthesis inhibition activity of the recombinant OsRIP1 and controls (DPBS buffer and BSA) was determined using an in vitro transcription/translation system [49 (link)]. To test the effect of OsRIP1 on animal ribosomes the TnT^® T7 Quick Coupled Transcription/Translation System Kit (Promega, Mannheim, Germany) based
On a cell-free system derived from rabbit reticulocytes. The effect on plant ribosomes was analyzed using the TnT^® T7 Coupled Wheat Germ Extract kit (Promega) was used. According to the manufacturer’s instructions, the prepared mixture was incubated at 30°C for 10 min and chilled on ice. Afterwards, 2 µL DPBS buffer or buffer containing different concentrations of OsRIP1 was added to the reaction mixture and incubated for 30 min at 30 °C. After addition of 35 µL nuclease-free water at room temperature, the reaction samples were transferred to a luminometer plate (Greiner Labortechnik, Frickenhausen, Germany) containing 5 µL luciferase assay reagent at 25 °C. The relative luciferase activities of the samples were determined using a microtiter top plate reader (Infinite F200 Pro, Tecan, Mannedorf, Switzerland).