Cells were lysed in 20 mM Hepes buffer, 10 mM KCl, 1 mM EDTA, 0.2% NP-40, 10% Glycerol44 (link), 59 (link) and co-immunoprecipitation studies were performed as previously described.44 (link) Details can be found in Supplementary Information . The following antibodies were used: mouse monoclonal anti-β-Actin (AC-15, Sigma-Aldrich, St Louis, MO, USA), mouse anti-HSP90 (Heat Shock Protein 90), rabbit polyclonal anti-KLF4 (sc-20691), rabbit polyclonal anti-p21 (C-19; sc-397), mouse monoclonal anti-p53 (DO-1; sc-126), mouse anti-Myc (9E10), mouse monoclonal anti-Caspase-3 (E-8; sc-7272), goat anti-Fibrillarin (D-14; sc-11336), goat anti-GAPDH (V-18; sc-20357), and mouse anti-BRAF wt (sc-5284) (Santa Cruz Biotechnology), rabbit polyclonal anti-BAX, rabbit polyclonal anti-BCL2, rabbit polyclonal anti-E2F1, rabbit polyclonal anti-poly ADP-ribose polymerase, rabbit polyclonal anti-Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), rabbit polyclonal anti-Cleaved Caspase 3 (Asp175) (Cell Signaling Technologies, Danvers, MA, USA). Chemiluminescent detection was used.
Protocol detail
Co-immunoprecipitation Assay for Protein-Protein Interactions
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anti-β-Actin (AC-15,
mouse anti-Myc (9E10),
goat anti-Fibrillarin (D-14;
goat anti-GAPDH (V-18;
anti-Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204),
anti-Cleaved Caspase 3 (Asp175)
Actin
Antibodies
Bcl2
Braf
Buffer
Caspase 3
Cells
Co immunoprecipitation
E2f1
Edta
Erk1
Fibrillarin
Gapdh
Goat
Heat shock protein 90
Hepes
Klf4
Mouse
Mouse caspase 3
Np 40
Poly adp ribose polymerase
Rabbit
Corresponding Organization : Azienda Ospedaliero-Universitaria Careggi
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Variable analysis
independent variables
- Lysis buffer composition: 20 mM Hepes, 10 mM KCl, 1 mM EDTA, 0.2% NP-40, 10% Glycerol
- Protein-protein interactions (co-immunoprecipitation studies)
- Antibodies used for immunoprecipitation and detection: mouse monoclonal anti-β-Actin, mouse anti-HSP90, rabbit polyclonal anti-KLF4, rabbit polyclonal anti-p21, mouse monoclonal anti-p53, mouse anti-Myc, mouse monoclonal anti-Caspase-3, goat anti-Fibrillarin, goat anti-GAPDH, and mouse anti-BRAF wt, rabbit polyclonal anti-BAX, rabbit polyclonal anti-BCL2, rabbit polyclonal anti-E2F1, rabbit polyclonal anti-poly ADP-ribose polymerase, rabbit polyclonal anti-Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), rabbit polyclonal anti-Cleaved Caspase 3 (Asp175)
- No positive or negative controls were explicitly mentioned in the provided information.
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