TDRKH-AS1 and its antisense RNA were made from transcription with the Biotin RNA Labeling Mix (Roche, USA) and T7 RNA polymerase (Roche, USA). Biotinylated RNA was incubated with HUVECs nuclear extracts, and pulldown proteins were run on SDS-PAGE gels (Sigma, USA) and stained with silver staining solution (Beyotime, China) as our previous work [31 (link)]. The lncRNA pulled-down complex was sent to the company (Wininnovate Bio, China), using mass spectrometry to detect proteins interacting with TDRKH-AS1. We determined the proteins interacting with TDRKH-AS1, satisfying the criteria: only exists in the sense probe pulled-down complex and with at least 3 peptides. KEGG pathway enrichment analysis and GO (Gene Ontology) enrichment analysis were performed by clusterProfiler.
Silver staining solution
Manufactured by Beyotime
Sourced in China
Silver staining solution is a laboratory reagent used in various analytical techniques. It is a clear, aqueous solution that contains silver ions, which can be used to detect and visualize proteins, nucleic acids, or other biomolecules in gel electrophoresis or blotting procedures.
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4 protocols using silver staining solution
1
Identification of TDRKH-AS1 Interacting Proteins
2
SH3PXD2A-AS1 Interactome Profiling
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SH3PXD2A-AS1 and its antisense RNA were made from transcription with the Biotin RNA Labeling Mix (Roche, USA) and T7 RNA polymerase (Roche, USA). Biotinylated RNA was incubated with HTR8/SVneo cell nuclear extracts, and pulldown proteins were run on SDS-PAGE gels (Sigma, USA) and stained with silver staining solution (Beyotime, China), followed by Mass spectrometry.
3
Identification of INHBA-AS1 Interactors
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INHBA-AS1 and its antisense RNA were transcribed with the biotin RNA labeling mix (Roche, USA) and T7 RNA polymerase (Roche, USA). Biotinylated RNA was incubated with HTR8/SVneo cell nuclear extracts. The pulldown proteins were run on SDS-PAGE gels (Sigma, USA) and stained with silver staining solution (Beyotime, China), followed by mass spectrometry.
4
RNA Pull-Down Assay for Protein Interactors
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In RNA pull-down assays, PDIA3P1 RNA and its antisense RNA were transcribed and biotinylated in vitro using a DNA template with the T7 promoter, following the Ribo™ RNAmax-T7 Biotin Labeling Transcription Kit instructions (Cat. AM1344, Invitrogen, CA, USA). Biotinylated RNA was captured using streptavidin magnetic beads and then incubated with HTR-8/SVneo cell extract. The eluted protein from the RNA–protein complex underwent sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), western blotting using HuR antibody, and staining with silver staining solution (Beyotime, China) for mass spectrometry. After elution of lncRNA-interacting proteins, mass spectrometric analysis was performed using an LTQ linear ion trap mass spectrometer (Thermo Finnigan, San Jose, CA). The detailed process of the RNA pull-down assay is listed in Supplementary 5 .
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