Cells were resuspended in RIPA buffer: 150 mM sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0, 1 mM dithiothreitol, 0.5 mM NaVO4, and protease inhibitor cocktail (Sigma-Aldrich). After 10 min on ice, extracts were centrifuged for 10 min at 12,000 g ,supernatants were collected and used for western blot as described (49 (link)). Primary antibody incubation (1:1000) was carried out with the following antibodies: PKM1, hnRNPA1, hnRNPA2/B1, hnRNPC1/C2 (Sigma-Aldrich); PKM2 (Cell Signaling Technology, MA, USA); PTBP1, SRSF1, SRP20, SRp40/p55/p75 (Santa Cruz Biotechnology, CA, USA); hnRNPF/H (Abcam, UK). PTBP2 antibody was a generous gift of Professor Douglas L. Black (UCLA, CA). Images of the western blot were acquired as TIFF files.
Protocol detail
Protein Extraction and Western Blot
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Antibodies
Antibody
Buffer
Cells
Dithiothreitol
Np 40
Protease inhibitor
Ripa
Sodium chloride
Sodium deoxycholate
Tris
Western blot
Corresponding Organization :
Other organizations : University of Rome Tor Vergata, Sapienza University of Rome, Fondazione Santa Lucia, Philipps University of Marburg
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Variable analysis
independent variables
- RIPA buffer composition (150 mM sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0, 1 mM dithiothreitol, 0.5 mM NaVO4, and protease inhibitor cocktail)
- Protein expression levels of PKM1, hnRNPA1, hnRNPA2/B1, hnRNPC1/C2, PKM2, PTBP1, SRSF1, SRP20, SRp40/p55/p75, hnRNPF/H, and PTBP2 as measured by Western blot
- Incubation time on ice (10 min)
- Centrifugation time and speed (10 min at 12,000 g)
- None specified
- None specified
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