Fig. 10. Cx43 phosphorylation is increased in adherent neurospheres. Cell lysates were prepared from either floating (grown for 10 days) or adherent (8 days) neurospheres. Equal amounts of proteins were incubated overnight at 37°C in the presence of either digestion buffer (lanes 1 and 4), alkaline phosphatase (lanes 2 and 5) or alkaline phosphatase plus an excess of phosphatase inhibitors (lanes 3 and 6). Samples were separated by SDS-gel electrophoresis and immunoblotted with either a rabbit polyclonal (A) or a mouse monoclonal (B) anti-Cx43 antibody. Both antibodies reacted with Cx43 from floating neurospheres, and the apparent electrophoretic mobility was not perturbed by alkaline phosphatase treatment (lanes 1-3). In adherent neurospheres, a broader band was detected when the blot was probed with the polyclonal antibody (panel A, lane 4). Following exposure to phosphatase, Cx43 shifted to faster migrating forms (panel A, lane 5). The monoclonal anti-Cx43 antibody, which recognizes non-phosphorylated Cx43, detected Cx43 species only when the lysates were treated with alkaline phosphatase (panel B, lane 5). It should be noted that twice the amount of protein was loaded in B. This blot is representative of three others from two independent experiments.