RNA probes of fragments located in the intron 10 or intron 11 of cortactin gene were prepared from pGEM-3zf vector. The primers are shown in Supplemental Table 5 . Cell extracts were isolated from HCT-116 cells using nuclear protein extraction reagents (Thermo Scientific). The reaction mixture contained 150 mg of the cell extracts and 2μg of each biotinylated RNA probe. RNA affinity capture was subsequently conducted with streptavidin-sepharose beads as described previously [50 ].
Nuclear protein extraction reagents
Manufactured by Thermo Fisher Scientific
Sourced in United States
Nuclear protein extraction reagents are specialized biochemical tools used to isolate and purify nuclear proteins from cells or tissue samples. These reagents facilitate the efficient extraction and separation of nuclear proteins, which are essential for various cellular processes and gene regulation.
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Lab products found in correlation
3 protocols using nuclear protein extraction reagents
1
Cortactin Gene Intron RNA Probes
2
RNA-Protein Interaction Identification
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Fragments located in the 3′ UTR of ARHGDIA were cloned into a pGEM-3zf (+) vector containing a T7 promoter. The primers are shown in Supplemental Table 2 . The biotin-labeled sense RNA probes were synthesized in vitro using T7 RNA polymerase (TaKaRa Bio). Cytoplasmic cell extracts were isolated from T98G cells using nuclear protein extraction reagents (Thermo Scientific). RNA affinity capture was subsequently conducted with streptavidin-Sepharose beads as described previously [16 (link)].
3
Identifying Protein Interactions with EV71 IRES
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T98G, HA, RD, and SH-SY5Y cells were pelleted, and then prepared using nuclear protein extraction reagents (Thermo, USA). A Bio-Rad protein assay was used to determine the protein concentrations of cell extracts. The RNA pull-down assay protocol was modified from method previously reported (Lin et al. 2008 (link)). Biotinylated EV71 IRES was used to capture interacting proteins. Bands of interest were excised, digested in gel with trypsin, and identified by Bruker Ultraflex matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).
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