Immunoprecipitation of Flag- and HA-tagged Proteins

The process of Immunoprecipitation was previously described^{44 (link)}. Briefly, Xenopus embryo lysates were prepared with ice-cold TNSG buffer (20 mM Tris–HCl pH 7.5, 137 mM NaCl and 1% NP-40). IPs were performed for 4 h with 25 embryo equivalent extracts using monoclonal Anti-V5-agarose (Sigma-Aldrich). Western blot analysis was performed using anti-Flag–horseradish peroxidase (HRP)-conjugated (1:5,000, Sigma-Aldrich), anti-HA–HRP-conjugated (1:5,000, Sigma-Aldrich) anti-V5–HRP-conjugated (1:5,000, Sigma-Aldrich), anti-Phospho-p44/42 MAPK (Erk1/2) (Cell Signaling Technology), and anti-p44/42 MAPK (Erk1/2) (Cell Signaling Technology) antibodies.