Gs gene linker

Cell lysate after treatment was prepared using Signosis nuclear extraction kit (SK-0001). Equal amount of nuclear lysates from treated or untreated cells were mixed with biotinylated STAT3 or NFκB probes from Signosis EMSA assay kit. After 30 mins incubation at 22° C, samples Were run on a 6.6% polyacrylamide gel using 0.5x Tris Borate buffer (TBE). Samples with bound probes Were transferred onto a nitrocellulose membrane. The membrane subjected to UV irradiation On a Biorad GS gene linker with 125 milli Joules for 2 minutes. Immobilized probes were incubated With Steptavidin-HRP and were detected suing chemiluminiscence substrate. Image was obtained on a Licor Odyssey FC machine.

In GTP-binding assays^{19 (link)}, increasing concentrations (from 31.25 nM to 2 μM) of recombinant GST-RagD variants were incubated with 1 nM α-³²P-GTP (Hartmann Analytics) for 4 h at 4 °C in 50 mM HEPES (pH 7.5), 100 mM potassium acetate, 2 mM MgCl₂, 2 mM DTT, and 0.1% CHAPS. The samples were then spotted onto a chilled metal block covered with Parafilm and to induce crosslinking, UV light of a total energy of 300 mJ was applied using GS Gene Linker (Bio-Rad). The samples were then mixed with Laemmli buffer, denatured for 10 min at 65 °C, and run on 7.5% SDS-PAGE gels that were then dried and analyzed by autoradiography.

For RAP-MS experiments, around 480 million A549^ACE2 or Huh-7 cells were infected with SARS-CoV-2 at MOI 5 PFU/cell. At 24 h post-infection, cells were washed with cold PBS and irradiated with 254 nm UV light on ice at a total dose of 800 mJ/cm² in a GS Gene Linker (Bio-Rad Laboratories). Cells were lysed in 1 ml RAP lysis buffer (10 mM Tris pH 7.5, 500 mM LiCl, 0.5% dodecyl maltoside (DDM), 0.5% sodium dodecyl sulphate (SDS), 0.1% sodium deoxycholate, EDTA-free Protease Inhibitor Cocktail (Sigma Aldrich, 11873580001) and 300 U/ml Murine RNase Inhibitor (NEB, M0314L)) per 10 million cells, collected by scraping, and lysates were stored at -80°C.

Rosette leaves were cut with forceps and transferred to six-well tissue culture plates containing 3 ml dH₂O. They were subsequently incubated for 24 h in a 23 or 37°C growth cabinet with otherwise standard conditions. For molecular analysis, 9 to 12 rosette leaves from 3 to 4 seedlings were pooled for heat or control treatment. Rosette leaves were then dried on absorbent paper, flash-frozen in liquid nitrogen, and stored at −80°C or directly processed.
For survival assays, seeds were sowed in vitro, stratified for 72 h in the dark at 4°C and grown 7 d in standard conditions before heat or UV treatment. Heat stress was applied for 24 h or 48 h. UV-irradiation was performed in an Et-OH sterilized UV chamber (GS Gene Linker; Bio-Rad) equipped with 254 nm bulbs. Plate lids were removed before irradiation at 10,000 J/m² and placed back immediately. Irradiated seedlings were transferred to a dark growth cabinet with standard conditions for 24 h to block photoreactivation before recovering in light for 5 d.

A mixture (8 μl) containing RnaG120 (0.5 pmol) and the [³²P]-labeled G+1H oligonucleotide (2 pmol) was heated at 90°C for 1 min to denature RNA. Annealing of the primer with RnaG120 was carried out at 32°C for 5 min in buffer A (see above) in the presence or absence of VirF. The mixture was transferred to an ice-cold plate and U.V. irradiated for 1 min using the GS Gene-linker BioRad (180 mJ, 254 nm bulbs at ∼12 cm from the U.V. source). Finally the cross-linked RNA was primer-extended using the AMV Reverse Transcriptase as previously described (Giangrossi et al., 2010 (link)).