Anti flag ha

Co-IP and western blotting were performed as described previously (62 (link)). Briefly, the CAB or 293T cells were seeded into 10 cm² dishes overnight, and transfected with different plasmids (a total of 10 μg) indicated on the Figure using FuGENE HD Transfection Reagent (Promega). At 24 h post-transfection, the cells were lysed by radioimmunoprecipitation (RIPA) lysis buffer with protease inhibitor cocktail (Sigma-Aldrich). After removing cellular debris, the supernatants were incubated with 15 μl anti-Flag or anti-Myc affinity gel (Sigma-Aldrich) overnight at 4°C. Immunoprecipitated proteins were resuspended in 100 μl SDS-PAGE protein loading buffer (Beyotime) after collecting by centrifugation and washing three times with lysis buffer. The whole cell lysates (WCL) or immunoprecipitated proteins were separated by 10–15% SDS-PAGE and then transferred to polyvinylidene fluoride membranes (Bio-Rad). The membranes were incubated with anti-β-actin (Cell Signaling Technology) at 1:3,000, anti-Flag/HA (Sigma-Aldrich) at 1:3,000, or anti-Myc (Santa Cruz Biotechnology) at 1:3,000, and then hybridized with the secondary HRP-conjugated anti-mouse IgG or anti-rabbit IgG (Thermo Scientific) at 1:5,000. Results are captured by using an ImageQuant LAS 4000 system (GE Healthcare) and are representative of three independent experiments.

Xenopus embryo lysates or LS174T cell (ATCC) 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 8 h with 25 embryo equivalent extracts using monoclonal Anti-Flag, HA or V5-agarose (Sigma-Aldrich). Endogenous protein IPs were conducted for 8 h with LS174T cell extracts using an anti-ephrinB2 antibody (1:500, C-18, Santa Cruz Biotechnology) or anti-Dsh2 antibodies (1:500, 3216, Cell Signaling). Western blot analysis was performed using anti-Flag–horseradish peroxidase (HRP)-conjugated (1:5000, A8592, Sigma), anti-HA–HRP-conjugated (1:5000, 12013819001, Roche), anti-phosphotyrosine–HRP-conjugated (1:1000, 4G10, Upstate Biotechnology), anti-ephrinB2 (1:1000, SAB4300456, sigma), and anti-ERK2 (1:1000, Santa Cruz Biotechnology) antibodies.

Immunoprecipitates or whole cell lysates were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane (Trans-Blot Turbo™ Transfer System, Bio-Rad). The membranes were blocked for 1 h at room temperature in TBST buffer (25 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20, pH 7.5) containing 5% nonfat dry milk, probed with the indicated primary Abs at an appropriate dilution overnight at 4°C, washed three times with TBST, and then incubated with secondary Abs for 1 h at room temperature. After three additional washes with TBST, the membranes were stained with the Immobilon Western chemiluminescent horseradish peroxidase (HRP) substrate (Millipore) and detected by using an ImageQuant LAS 4000 system (GE Healthcare). Abs were diluted as follows: anti-β-actin (Cell Signaling Technology) at 1:1,000, anti-Flag/HA (Sigma-Aldrich) at 1:3,000, anti-Myc (Santa Cruz Biotechnology) at 1:2,000, and HRP-conjugated anti-mouse IgG (Thermo Scientific) at 1:5,000. Results are representative of three independent experiments.