Coimmunoprecipitation and pulldown experiments.

A. HEK293T cells were cotransfected with plasmids to express GST-DCC fusion constructs and 3X-Flag-tagged B23 protein. Coimmunoprecipitation experiments were conducted with an anti-Flag antibody. GST pulldown, using GSH affinity resin, was performed in parallel. B. HEK293T cells were transfected with GST-DCC fusion protein expression plasmids. GST pulldown, using GSH affinity resin, was performed. Samples were analyzed by western blotting with antibodies to detect B23 (top panel) and I2PP2A (middle panel). C. HEK293T cells were cotransfected with plasmids to express GST-DCC fusion proteins and I2PP2A-Flag-HA. Immunoprecipitation was conducted with a mixture of antibodies against Flag and HA. Samples were analyzed by western blotting with antibodies to detect I2PP2A (top panel) and GST (bottom panel). LC, light chain.