Figure 6.

Interactions between SplitΔR fragments and R domain. (A) Co-immunoprecipitation of the front and back halves of CFTR: lane 1, in the absence of GST-R; lane 2, with unphosphorylated GST-R added to the lysate; lane 3, after addition of phosphorylated GST-R without phosphatase inhibitors; and lane 4, with phosphorylated GST-R added to lysates along with excess PKA, ATP, and phosphatase inhibitors (see Materials and methods). (B) Effect of PKA phosphorylation on electrophoretic mobility of GST-R. (a) Autoradiogram of GST after preincubation with PKA and [γ-³²P]ATP. (b–d) Western blots probed using anti-R domain mAb 25 (b), anti-R domain mAb 450 (c), or anti-GST antibody (d). (e) Unphosphorylated and phosphorylated GST-R in a Coomassie-stained gel. (C) Immunoprecipitates obtained with antibodies against the front or back half of SplitΔR after preincubation of lysates as described in (A). Note that GST-R was only detected when prephosphorylated. Electrophoretic mobility shifts confirmed that elevated phosphorylation promotes the interaction of GST-R, particularly with the amino-terminal half-molecule. (D) Co-immunoprecipitation of the front and back halves of CFTR from cells coexpressing SplitΔR_pIND+RD_pNUT CFTR. Phosphorylation was varied by pretreating cells for 3 h with H7 (lane 1), control medium (lane 2), or medium containing 150 μM cpt-cAMP+1 mM IBMX (lane 3). (E) Co-immunoprecipitation of endogenously expressed R domain with front or back halves of CFTR under the three conditions described in (D) except for lane 1 of the right panel, in which cells were pretreated with the PKA inhibitor H89. More R domain was pulled down with both half-molecules under phosphorylating conditions.