Uv stratalinker 1800

To examine the effects of TFEB gene regulatory variants on the binding sites for transcription factors, electrophoretic mobility shift assay (EMSA) was conducted using the LightShift^® Chemiluminescent EMSA kit (Thermo Fisher Scientific, Inc., Waltham, MA, United States). Biotinylated double-stranded oligonucleotides (30 bp) containing regulatory variants were used as probes. Nuclear extracts from HEK-293 and H9c2 cells were prepared using NE-PER^® Nuclear and Cytoplasmic Extraction Reagent kit (Thermo Fisher Scientific, Inc., Waltham, MA, United States). Protein concentrations were determined using the Bradford protein assay. DNA-protein binding reactions were conducted for 20 min at room temperature with equal amounts of probes (0.2 pMol) and nuclear extracts (3.0 μg). The reaction mixtures were subsequently separated on a 6% polyacrylamide gel and transferred onto a nylon membrane (Thermo Fisher Scientific, Inc., Waltham, MA, United States). The oligonucleotides were cross-linked to the membrane using the UV Stratalinker 1800 (Stratagene; Agilent Technologies, Inc., Santa Clara, CA, United States) and were detected by chemiluminescence using the LightShift^® Chemiluminescent EMSA kit (Thermo Fisher Scientific, Inc., Waltham, MA, United States).

AGO-expressing 293 cells, mock-infected or infected with RSV at MOI of 1. At 15 h p.i., the total cell lysates were prepared after the UV crosslinking (UV Stratalinker 1800, Stratagene/Agilent, Santa Clara, CA, USA) and harvested using cell lysis buffer (IP assay kit, Roche, Indianapolis, IN, USA) in the presence of RNase inhibitors. Samples were pre-cleared using protein A/G agarose beads and incubated with an anti-Flag antibody for 1 h at 4 °C. After the addition of protein A/G agarose beads, samples were incubated overnight at 4 °C. The IP complexes were washed using buffers provided by the kit and then treated with proteinase K (New England BioLabs, Ipswich, MA, USA), followed by RNA extraction using Trizol. Associated RNAs in Flag-tagged AGO complexes were analyzed by NB for tRF5-GluCTC. Small amounts from the IP complexes were aliquoted and followed by Western blot using anti-RSV antibodies. The membrane was stripped and reprobed with anti-Flag antibodies to ensure the proper IP process.

Cells from each cell line were plated in quadruplicate at 3×10⁵ cells/well in 6 well plates and grown for two days. Two wells of each cell line were exposed to 90 mJ/cm² 254 nm UV radiation in a UV Stratalinker 1800 (Agilent Technologies, Santa Clara, CA). Cells were harvested 20 hours later using Tryple (Life Technologies), rinsed in PBS and Caspase activity measured using the EnzCheck Caspase-3 Assay Kit #1 as per instructions (Molecular Probes, Eugene, USA).

Electrophoretic mobility shift assay (EMSA) was performed with the LightShift® Chemiluminescent EMSA kit (Thermo Fisher Scientific) according to the procedure. H9c2 cell nuclear extracts were prepared with NE-PER® Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific). Biotinylated double-stranded oligonucleotides (30 bp) with or without the variants were used as probes. The DNA–protein reactions were incubated for 20 min at room temperature. The reaction mixtures were separated on a 6% polyacrylamide gel, and subsequently transferred onto a nylon membrane (Thermo Fisher Scientific). Oligonucleotides were cross-linked using the UV Stratalinker 1800 (Agilent Technologies, Inc., Santa Clara, CA, USA). Signals were examined by chemiluminescence.