Figure 5.

Autoradiographic analysis of PKC-mediated phosphorylation of the K_ATP channel. (A) Autoradiograph showing an assay of PKC-catalyzed phosphorylation of the band corresponding to antibody-purified ΔC26/FLAG proteins with either threonine (T) or alanine (A) at position 180 (lanes 1 and 2). Phosphorylation of membrane proteins from tsA201 cells expressing either ΔC26/FLAG or ΔC26(T180A)/FLAG was performed by using [γ-³²P]ATP as the phosphate donor. The PKC concentration in the reaction mixture was 250 nM. For the indicated bands, the PKC inhibitor PKC(19–31) (500 nM) or chelerythrine (5 μM) was added to the phosphorylation reaction mixture before the addition of PKC. Under these conditions, the specific inhibitor peptide PKC(19–31) (lanes 3 and 4) caused a significant reduction in phosphorylation (see Materials and Methods, Phosphorylation Assays section) whereas chelerythrine (lanes 5 and 6) almost completely prevented PKC-catalyzed phosphorylation. (B) Corresponding Western blot of the same protein samples as in A, indicating comparable levels of ΔC26/FLAG protein in all lanes.