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. 2018 Jun 4;217(6):2167–2184. doi: 10.1083/jcb.201708053

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© 2018 Isensee et al.

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Figure 5. — Presence of cAMP increases the binding of pRII antibodies to PKA-II holoenzymes. (A–D) SPR analysis using pRII- (A-B) or RRXpS/pT-specific antibodies (C and D) covalently immobilized on sensor chips. Binding of preformed RIIα₂:Cα₂ (A and C) and RIIβ₂:Cα₂ holoenzymes (B and D) was assessed in buffer containing 1 mM MgCl₂ or 1 mM CaCl₂ and 0.5 mM ATP. Dissociation of holoenzymes was induced by adding 10 µM cAMP before SPR analysis. (E) Coimmunoprecipitation of RIIβ and Cα using pRII antibodies captured on protein G functionalized beads. Pull-downs of preformed RIIβ₂:C₂ holoenzymes were performed in the absence or presence of 1 mM ATP or 0.5 and 10 µM cAMP. Representative immunoblots (left) and densitometry results are shown (right). Values are means ± SD; n = 3. IP, immunoprecipitation; RU, resonance units.