Agarose beads sc 2003

For co-IPs of exogenous protein interactions, the injected embryos were lysed with TNSG buffer including protease inhibitors. Tagged proteins were precipitated with monoclonal anti-HA agarose beads (A2095; Sigma-Aldrich), anti-V5 agarose beads (A7345; Sigma-Aldrich), or anti-Flag M2 affinity gel (A2220; Sigma-Aldrich). For GFP-tagged protein pull-down, anti-GFP antibody was used. The resultant immunocomplexes were formed for 8 h at 4°C on a nutator and washed with lysis buffer four times. For co-IPs of endogenous protein interactions, HT29 and LS174T cells were lysed with TNSG containing 0.5% NP-40, and 10 µl of anti-Dvl2 antibody was incubated with 2 mg of total lysates at 4°C overnight, followed by an additional incubation with protein A/G plus agarose beads (sc-2003; Santa Cruz Biotechnology) for 2 h. Bead immunocomplexes were washed three times with the same lysis buffer. To examine coprecipitation of Drg1 with Dvl2, IB with Drg1 antibody was followed.

Cleared whole cell or cell fraction protein lysates were incubated at 4°C for 3 hours with the appropriate antibody precoupled to protein A/G plus agarose beads (sc-2003, Santa Cruz Biotechnology). The beads were washed twice with extraction buffer, twice with extraction buffer containing 0.5 M LiCl, and twice with assay buffer [40 mM tris-Cl (pH 7.5), 0.1 mM EDTA, 5 mM MgCl₂, and 2 mM DTT].

For immunoprecipitation experiments, cortical fiber portions of embryonic day 19 chick lenses were collected. CEF cells were coinfected with recombinant retroviruses containing WT Cx50 and Cx50 truncation mutants: Cx50(368T) and Cx50(379T). Crude membranes were prepared similarly as described above (Western blotting), while the pellets were resuspended in immunoprecipitation buffer (100 mM NaCl, 15 mM EDTA, 20 mM Na₂B₄O₇, and 0.5% Triton X-100, pH 8.5) for coimmunoprecipitation assays. Lysates (120 µg per sample) were precleared with protein A/G plus agarose beads (sc2003; Santa Cruz) and pelleted by centrifugation at 1,000 g at 4°C for 5 min. Supernatants (precleared lysates) were then incubated with anti-Cx50 CT, anti-Cx50 Loop (against intracellular loop domain of Cx50), anti-FLAG tag, or rabbit IgG antibody at 4°C overnight, followed by precipitation of the complexes after incubation with 120 µl of protein A/G plus agarose beads for 2 h at room temperature. Immunoprecipitates were collected in reducing or nonreducing sample buffer for detection of integrin α6 or Cx50, respectively, separated on 10% SDS-PAGE gels, and immunoblotted with anti-Cx50 antibody or anti-integrin α6 antibody, respectively.