Figure 5.

Absence of phosphotyrosines (P-Tyr) in the channel subunits. αβγ MDCK cells were labeled with [³⁵S]methionine and treated either with aldosterone for 16 h or with insulin for 10 min. The subunits were recovered by anti-α, -β, and -γ sera. The products of the first immunoprecipitation were subjected to a second round of immunoprecipitation with an antiphosphotyrosine antibody (lanes 1, 5, and 9). The channel subunits not bound by the antiphosphotyrosine antibody were reprecipitated with specific subunit antibodies (lanes 2–4, 6–8, and 10–12). Lane 14 shows all proteins immunoprecipitated with the antiphosphotyrosine antibody from a lysate of insulin-stimulated cells. Bars indicate molecular masses of 85 and 45 kDa, respectively.